Graphitic nanotubes in luminescence assays

ABSTRACT

Graphitic nanotubes, which include tubular fullerenes (commonly called &#34;buckytubes&#34;) and fibrils, which are functionalized by chemical substitution, are used as solid supports in electrogenerated chemiluminescence assays. The graphitic nanotubes are chemically modified with functional group biomolecules prior to use in an assay. Association of electrochemiluminescent ruthenium complexes with the functional group biomolecule-modified nanotubes permits detection of molecules including nucleic acids, antigens, enzymes, and enzyme substrates by multiple formats.

This application is a continuation-in-part of copending Fischer et al.application Ser. No. 08/352,400 entitled Functionalized Nanotubes, filedDec. 8, 1994. The subject matter of this application is incorporated byreference.

FIELD OF THE INVENTION

This application relates generally to methods and apparatus forconducting binding assays, more particularly to those which measure thepresence of an analyte of interest by measuring luminescence emitted byone or more labeled components of the assay system. More specifically,the invention relates to precise, reproducible, accurate homogeneous orheterogeneous specific binding assays of improved sensitivity in whichthe luminescent component is concentrated in the assay composition andcollected on the detection system before being caused toelectrochemiluminescence.

BACKGROUND OF THE INVENTION

Numerous methods and systems have been developed for the detection andquantitation of analytes of interest in biochemical and biologicalsubstances. Methods and systems which are capable of measuring traceamounts of microorganisms, pharmaceuticals, hormones, viruses,antibodies, nucleic acids and other proteins are of great value toresearchers and clinicians.

A very substantial body of art has been developed based upon the wellknown binding reactions, e.g., antigen-antibody reactions, nucleic acidhybridization techniques, and protein-ligand systems. The high degree ofspecificity in many biochemical and biological binding systems has ledto many assay methods and systems of value in research and diagnostics.Typically, the existence of an analyte of interest is indicated by thepresence or absence of an observable "label" attached to one or more ofthe binding materials. Of particular interest are labels which can bemade to luminesce through photochemical, chemical, and electrochemicalmeans. "Photoluminescence" is the process whereby a material is inducedto luminesce when it absorbs electromagnetic radiation. Fluorescence andphosphorescence are types of photoluminescence. "Chemiluminescent"processes entail the creation of luminescent species by chemicaltransfer of energy. "Electrochemiluminescence" entails creation ofluminescent species electrochemically.

Chemiluminescent assay techniques where a sample containing an analyteof interest is mixed with a reactant labeled with a chemiluminescentlabel have been developed. The reactive mixture is incubated and someportion of the labeled reactant binds to the analyte. After incubation,the bound and unbound fractions of the mixture are separated and theconcentration of the label in either or both fractions can be determinedby chemiluminescent techniques. The level of chemiluminescencedetermined in one or both fractions indicates the amount of analyte ofinterest in the biological sample.

Electrochemiluminescent (ECL) assay techniques are an improvement onchemiluminescent techniques. They provide a sensitive and precisemeasurement of the presence and concentration of an analyte of interest.In such techniques, the incubated sample is exposed to a voltammetricworking electrode in order to trigger luminescence. In the properchemical environment, such electrochemiluminescence is triggered by avoltage impressed on the working electrode at a particular time and in aparticular manner. The light produced by the label is measured andindicates the presence or quantity of the analyte. For a fullerdescription of such ECL techniques, reference is made to PCT publishedapplication US85/01253 (WO86/02734), PCT published application numberUS87/00987, and PCT published application U.S. 88/03947. The disclosuresof the aforesaid applications are incorporated by reference.

It is desirable to carry out electrochemiluminescent assays without theneed for a separation step during the assay procedure and to maximizethe signal modulation at different concentrations of analyte so thatprecise and sensitive measurements can be made. Among prior art methodsfor nonseparation assays are those which employ microparticulate mattersuspended in the assay sample to bind one or more of the bindingcomponents of the assay.

U.S. application Ser. No. 539,389 (PCT published application U.S.89/04919) teaches sensitive, specific binding assay methods based on aluminescent phenomenon wherein inert microparticulate matter isspecifically bound to one of the binding reactants of the assay system.The assays may be performed in a heterogeneous (one or more separationsteps) assay format and may be used most advantageously in a homogeneous(nonseparation) assay format.

U.S. 89/04919 relates to a composition for an assay based upon a bindingreaction for the measurement of luminescent phenomenon, whichcomposition includes a plurality of suspended particles having a surfacecapable of binding to a component of the assay mixture. In anotheraspect, it is directed to a system for detecting or quantitating ananalyte of interest in a sample, which system is capable of conductingthe assay methods using the assay compositions of the inventions. Thesystem includes means for inducing the label compound in the assaymedium to luminesce, and means for measuring the luminescence to detectthe presence of the analyte of interest in the sample.

It was found that the binding of that component of the assay system towhich an electrochemiluminescent moiety has been linked, to suspendedmicroparticulate matter, greatly modulates the intensity of theluminescent signal generated by the electrochemiluminescent moietylinked to that component, thereby providing a means of monitoring thespecific binding reaction of the assay system. The suspended particleswere found to have little or no effect on the intensity of theluminescent signal generated by the electrochemiluminescent moietylinked to the component of the system which remains unbound to thesuspended microparticulate matter.

Thus, U.S. 89/04919 is directed to methods for the detection of ananalyte of interest in a sample, which method includes the steps of (1)forming a composition comprising (a) a sample suspected of containing ananalyte of interest, (b) an assay-performance-substance selected fromthe group consisting of (i) analyte of interest or analog of the analyteof interest, (ii) a binding partner of the analyte of interest or itssaid analog, and (iii) a reactive component capable of binding with (i)or (ii), wherein one of said substances is linked to a label compoundhaving a chemical moiety capable of being induced to luminesce, and (c)a plurality of suspended particles capable of specifically binding withthe analyte and/or a substance defined in (b)(i), (ii), or (iii); (2)incubating the composition to form a complex which includes a particleand said label compound; (3) inducing the label compound to luminesce;and (4) measuring the luminescence emitted by the composition to detectthe presence of the analyte of interest in the sample. Those samemethods may be used to quantify the amount of analyte in a sample bycomparing the luminescence of the assay composition to the luminescenceof a composition containing a known amount of analyte.

Analogs of the analyte of interest, which may be natural or synthetic,are compounds which have binding properties comparable to the analyte,but include compounds of higher or lower binding capability as well.Binding partners suitable for use in the present invention arewell-known. Examples are antibodies, enzymes, nucleic acids, lectins,cofactors and receptors. The reactive components capable of binding withthe analyte or its analog and/or with a binding partner thereof may be asecond antibody or a protein such as Protein A or Protein G or may beavidin or biotin or another component known in the art to enter intobinding reactions.

Advantageously, the luminescence arises from electrochemiluminescence(ECL) induced by exposing the label compound, whether bound or unboundto specific binding partners, to a voltammetric working electrode. TheECL reactive mixture is controllably triggered to emit light by avoltage impressed on the working electrode at a particular time and in aparticular manner to generate light. Although the emission of visiblelight is an advantageous feature the composition or system may emitother types of electromagnetic radiation, such as infrared orultraviolet light, X-rays, microwaves, etc. Use of the terms"electrochemiluminescence," "electrochemiluminescent""electrochemiluminescence" "luminescence," "luminescent," and"luminesce" includes the emission of light and other forms ofelectromagnetic radiation.

The methods taught in U.S. 89/04919 permit the detection andquantitation of extremely small quantities of analytes in a variety ofassays performed in research and clinical settings. The demands ofresearchers and clinicians makes it imperative, however, to lower thedetection limits of assays performed by these methods to increase thesensitivities of those assays and to increase the speed at which theycan be performed.

Various methods are known in the art for increasing the signal fromlabeled species by concentrating them before subjecting them to ameasurement step. In U.S. Pat. No. 4,652,333, for example, particleslabeled with fluorescent, phosphorescent or atomic fluorescent labelsare concentrated by microfiltration before a measurement step isperformed.

It is also known in the art to concentrate labeled immunochemicalspecies prior to a measurement step, by, e.g., drawing magneticallyresponsive labeled particles to the surface of a measurement vessel. InU.S. Pat. Nos. 4,731,337, 4,777,145, and 4,115,535, for example, suchparticles are drawn to the vessel wall and then are irradiated to excitea fluorophoric emission of light.

In U.S. Pat. No. 4,945,045, particles are concentrated on a magneticelectrode. An electrochemical reaction takes place at the electrodefacilitated by a labeled chemical mediator. The immunochemical bindingreaction alters the efficiency of the mediator resulting in a modulatedsignal when binding takes place.

While not being bound by any particular mechanistic explanation ofsurface excitation, e.g., electrochemiluminescence, it is believed thatthe label on the solid-phase complex must be oxidized at the electrode.This requires that an electron move from the label to the electrode. Itis believed that the electron makes this "jump" by a phenomenon known astunneling in which the electron passes through space (a region where itspotential energy is very high, e.g., the solution) without having to go"over" the potential energy barrier. It can tunnel through the energybarrier, and thus, move from one molecule to another or from onemolecule to an electrode without additional energy input. However, thistunneling phenomenon can only operate for very short distances. Theprobability of the tunneling phenomenon falls off exponentially as thedistance between the two species increases. The probability of thetunneling phenomenon occurring between two species is fairly high if thedistance is less than 25 Angstroms (2.5 nm) but is fairly low if thedistance is greater. The distance of 25 Å is a rule-of-thumb used bythose skilled in the art but is not an absolute limitation.

Accordingly, only those ECL labels with 25 Å of the surface of theelectrode can be expected to participate in the ECL process. The area ofthe particle which is within 25 Å of the surface of an electrode istypically extremely small.

Accordingly, one would not expect that ECL from a particle surface wouldbe measurable to any significant degree. Moreover, the light which isproduced by the ECL process must pass through the particle to get to thephotomultiplier. Since the particles are essentially opaque (aconcentrated suspension of them is black) one would not expect that,even if significant amounts of light could be produced by ECL, that thelight could pass through the particle and be measured by thephotomultiplier.

Since the 1970s graphitic nanotubes and fibrils have been identified asmaterials of interest for a variety of applications. Submicron graphiticfibrils are sometimes called vapor grown carbon fibers. Carbon fibrilsare vermicular carbon deposits having diameters less than 1.0μ,preferably less than 0.5μ, and even more preferably less than 0.2μ. Theyexist in a variety of forms and have been prepared through the catalyticdecomposition of various carbon-containing gases at metal surfaces. Suchvermicular carbon deposits have been observed almost since the advent ofelectron microscopy. A good early survey and reference is found in Bakerand Harris, Chemistry and Physics of Carbon, Walker and Thrower ed.,Vol. 14, 1978, p. 83, hereby incorporated by reference. See also,Rodriguez, N., J. Mater. Research, Vol. 8, p. 3233 (1993), herebyincorporated by reference.

In 1976, Endo et al. (see Obelin, A. and Endo, M., J. of Crystal Growth,Vol. 32 (1976), pp. 335-349, hereby incorporated by reference)elucidated the basic mechanism by which such carbon fibrils grow. Therewere seen to originate from a metal catalyst particle, which, in thepresence of a hydrocarbon containing gas, becomes supersaturated incarbon. A cylindrical ordered graphitic core is extruded whichimmediately, according to Endo et al., becomes coated with an outerlayer of pyrolytically deposited graphite. These fibrils with apyrolytic overcoat typically have diameters in excess of 0.1μ, moretypically 0.2 to 0.5μ.

In 1983, Tennent, U.S. Pat. No. 4,663,230, hereby incorporated byreference, succeeded in growing cylindrical ordered graphite cores,uncontaminated with pyrolytic carbon. Thus, the Tennent inventionprovided access to smaller diameter fibrils, typically 35 to 700 Å(0.0035 to 0.070μ) and to an ordered, "as grown" graphitic surface.Fibrillar carbons of less perfect structure, but also without apyrolytic carbon outer layer have also been grown.

Fibrils, buckytubes and nanofibers are distinguishable from continuouscarbon fibers commercially available as reinforcement materials. Incontrast to fibrils, which have, desirably large, but unavoidably finiteaspect ratios, continuous carbon fibers have aspect ratios (L/D) of atleast 10⁴ and often 10⁶ or more. The diameter of continuous fibers isalso far larger than that of fibrils, being always >1.0μ and typically 5to 7μ

Continuous carbon fibers are made by the pyrolysis of organic precursorfibers, usually rayon, polyacrylonitrile (PAN) and pitch. Thus, they mayinclude heteroatoms within their structure. The graphitic nature of "asmade" continuous carbon fibers varies, but they may be subjected to asubsequent graphitization step. Differences in degree of graphitization,orientation and crystallinity of graphite planes, if they are present,the potential presence of heteroatoms and even the absolute differencein substrate diameter make experience with continuous fibers poorpredictors of nanofiber chemistry.

Tennent, U.S. Pat. No. 4,663,230 describes carbon fibrils that are freeof a continuous thermal carbon overcoat and have multiple graphiticouter layers that are substantially parallel to the fibril axis. As suchthey may be characterized as having their c-axes, the axes which areperpendicular to the tangents of the curved layers of graphite,substantially perpendicular to their cylindrical axes. They generallyhave diameters no greater than 0.1μ and length to diameter ratios of atleast 5. Desirably they are substantially free of a continuous thermalcarbon overcoat, i.e., pyrolytically deposited carbon resulting fromthermal cracking of the gas feed used to prepare them.

Tennent, et al., U.S. Pat. No. 5,171,560, hereby incorporated byreference, describes carbon fibrils free of thermal overcoat and havinggraphitic layers substantially parallel to the fibril axes such that theprojection of said layers on said fibril axes extends for a distance ofat least two fibril diameters. Typically, such fibrils are substantiallycylindrical, graphitic nanotubes of substantially constant diameter andcomprise cylindrical graphitic sheets whose c-axes are substantiallyperpendicular to their cylindrical axis. They are substantially free ofpyrolytically deposited carbon, have a diameter less than 0.1μ and alength to diameter ratio of greater than 5. These fibrils are of primaryinterest in the invention.

Further details regarding the formation of carbon fibril aggregates maybe found in the disclosure of Snyder et al., U.S. patent applicationSer. No. 149,573, filed Jan. 28, 1988, and PCT Application No.US89/00322, filed Jan. 28, 1989 ("Carbon Fibrils") WO 89/07163, and Moyet al., U.S. patent application Ser. No. 413,837 filed Sep. 28, 1989 andPCT Application No. US90/05498, filed Sep. 27, 1990 ("Fibril Aggregatesand Method of Making Same") WO 91/05089, all of which are assigned tothe same assignee as the invention here and are hereby incorporated byreference.

Moy et al., U.S. Ser. No. 07/887,307 filed May 22, 1992, herebyincorporated by reference, describes fibrils prepared as aggregateshaving various macroscopic morphologies (as determined by scanningelectron microscopy) in which they are randomly entangled with eachother to form entangled balls of fibrils resembling bird nests ("BN");or as aggregates consisting of bundles of straight to slightly bent orkinked carbon fibrils having substantially the same relativeorientation, and having the appearance of combed yarn ("CY") e.g., thelongitudinal axis of each fibril (despite individual bends or kinks)extends in the same direction as that of the surrounding fibrils in thebundles; or, as, aggregates consisting of straight to slightly bent orkinked fibrils which are loosely entangled with each other to form an"open net" ("ON") structure. In open net structures the degree of fibrilentanglement is greater than observed in the combed yarn aggregates (inwhich the individual fibrils have substantially the same relativeorientation) but less than that of bird nests. CY and ON aggregates aremore readily dispersed than BN making them useful in compositefabrication where uniform properties throughout the structure aredesired.

When the projection of the graphitic layers on the fibril axis extendsfor a distance of less than two fibril diameters, the carbon planes ofthe graphitic nanofiber, in cross section, take on a herring boneappearance. These are termed fishbone fibrils. Geus, U.S. Pat. No.4,855,091, hereby incorporated by reference, provides a procedure forpreparation of fishbone fibrils substantially free of a pyrolyticovercoat. These fibrils are also useful in the practice of theinvention.

Carbon nanotubes of a morphology similar to the catalytically grownfibrils described above have been grown in a high temperature carbon arc(Iijima, Nature 354 56 1991). It is now generally accepted (Weaver,Science 265 1994) that these arc-grown nanofibers have the samemorphology as the earlier catalytically grown fibrils of Tennent. Arcgrown carbon nanofibers are also useful in the invention.

OBJECTS OF THE INVENTION

It is therefore an object of this invention to provide luminescenceassays using particles having a high surface area for immobilization ofassay performance substances to achieve advantageously high lightemission.

It is also an object of this invention to provide compositions andassays using graphitic nanotubes (fibrils) which can be labeled withcompounds capable of being induced to luminesce.

It is further object to provide functionalized fibrils for use in suchassays.

DESCRIPTION OF THE INVENTION Definition of Terms

The term "ECL moiety," "metal-containing ECL moiety" "label," "labelcompound," and "label substance," are used interchangeably. It is withinthe scope of the invention for the species termed "ECL moiety,""metal-containing ECL moiety," "organo-metallic," "metal chelate,""transition metal chelate" "rare earth metal chelate," "label compound,""label substance" and "label" to be linked to molecules such as ananalyte or an analog thereof, a binding partner of the analyte or ananalog thereof, and further binding partners of such aforementionedbinding partner, or a reactive component capable of binding with theanalyte, an analog thereof or a binding partner as mentioned above. Theabove-mentioned species can also be linked to a combination of one ormore binding partners and/or one or more reactive components.Additionally, the aforementioned species can also be linked to ananalyte or its analog bound to a binding partner, a reactive component,or a combination of one or more binding partners and/or one or morereactive components. It is also within the scope of the invention for aplurality of the aforementioned species to be bound directly, or throughother molecules as discussed above, to an analyte or its analog. Forpurposes of brevity, these ligands are referred to as anassay-performance-substance.

The terms detection and quantitation are referred to as "measurement",it being understood that quantitation may require preparation ofreference compositions and calibrations.

The terms collection and concentration of complex may be usedinterchangeably to describe the concentration of complex within theassay composition and the collection of complex at, e.g., an electrodesurface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a cell for performing themicroparticulate-based nonseparation and separation assays of theinvention.

FIG. 2 is a simplified diagram of a voltage control apparatus for usewith the cell of FIG. 1.

FIG. 3 is a schematic representation showing the specific enzymehydrolysis sites on Ru(bpy)₃ ²⁺ -labeled peptide fibrils.

FIG. 4 is a schematic representation of a DNA probe assay usingavidin-fibrils.

FIG. 5 is a schematic representation of a reaction scheme for thepreparation of bifunctional fibrils by the addition of N.sub.ε-(tert-butoxycarbonyl)-L-lysine.

FIG. 6 is a schematic representation of a reaction scheme forsynthesizing the ECL-based, bifunctional biosensor fibril.

FIG. 7 is a schematic representation of bifunctional ECL-based biosensorfibril.

FIG. 8 is a schematic representation of the reaction catalyzed byD-glucose-6-phosphate dehydrogenase.

FIG. 9 is a graph showing the ECL detection of glucose-6-phosphatedehydrogenase using a fibril-supported ECL-based biosensor.

FIG. 10 is a schematic representation of an alcohol dehydrogenase (ADH)based ECL biosensor.

FIG. 11 is a schematic representation of an ADH-based biosensorimmobilized on Dynal M450 beads (left) and immobilized on alkyl fibrils(right).

FIG. 12 is a graph showing ECL detection of ethanol using an enzymebiosensor immobilized on beads and fibrils.

FIG. 13 is a graphical representation of an assay of Ru(bpy)₃ ²⁺ bindingto carboxy fibrils and PEG-modified fibrils prepared by two differentmethods.

BRIEF DESCRIPTION OF THE INVENTION

In its broadest embodiment, the invention is in a nanotube to which isattached a component linked to a label compound capable of being inducedto luminesce. In particular, said nanotube is a graphitic nanotube andsaid luminescence is electrochemiluminescence. In one embodiment, saidcomponent is an enzyme biosensor.

The invention is also in a composition for the detection of an analyteof interest present in a sample, which composition comprises:

(i) a graphitic nanotube having a functional group and

(ii) an assay-performance-substance linked to said functional group,said assay-performance-substance being capable of binding, directly orindirectly, to the analyte.

In one embodiment, the assay-performance-substance being bound to saidfunctional group is in turn bound to the analyte. The compositionfurther comprises a second assay-performance-substance bound to theanalyte, said second assay-performance-substance being linked to a labelcompound capable of being induced to luminesce.

The assay-performance-substance contains at least one substance selectedfrom the group consisting of

(i) added analyte of interest or added analogue of said analyte;

(ii) a binding partner of said analyte or a binding partner of saidanalogue; and

(iii) a reactive component capable of binding with (i) or (ii).

One of said substances (i), (ii) or (iii) is linked to a label compoundhaving a chemical moiety capable of being induced to luminesce.

Broadly the particles are functionalized fibrils, i.e., fibrils whosesurface has been reacted or contacted with one or more substances toprovide active sites thereon for chemical substitution or physicaladsorption of different chemical species. McCarthy et al., U.S. patentapplication Ser. No. 351,967 filed May 15, 1989, hereby incorporated byreference, describes processes for oxidizing the surface of carbonfibrils that include contacting the fibrils with an oxidizing agent thatincludes sulfuric acid (H₂ SO₄) and potassium chlorate (KClO₃) underreaction conditions (e.g., time, temperature, and pressure) sufficientto oxidize the surface of the fibril. The fibrils oxidized according tothe processes of McCarthy, et al. are non-uniformly oxidized, that is,the carbon atoms are substituted with a mixture of carboxyl, aldehyde,ketone, phenolic and other carbonyl groups.

Fibrils have also been oxidized non-uniformly by treatment with nitricacid. International Application PCT/US94/10168 discloses the formationof oxidized fibrils containing a mixture of functional groups.Hoogenvaad, M. S., et al. ("Metal Catalysts supported on a Novel CarbonSupport", Presented at Sixth International Conference on ScientificBasis for the Preparation of Heterogeneous Catalysts, Brussels, Belgium,September 1994) also found it beneficial in the preparation offibril-supported precious metals to first oxidize the fibril surfacewith nitric acid. Such pretreatment with acid is a standard step in thepreparation of carbon-supported noble metal catalysts, where, given theusual sources of such carbon, it serves as much to clean the surface ofundesirable materials as to functionalize it.

In published work, McCarthy and Bening (Polymer Preprints ACS Div. ofPolymer Chem. 30 (1)420(1990)) prepared derivatives of oxidized fibrilsin order to demonstrate that the surface comprised a variety of oxidizedgroups. The compounds they prepared, phenylhydrazones,haloaromaticesters, thallous salts, etc., were selected because of theiranalytical utility, being, for example, brightly colored, or exhibitingsome other strong and easily identified and differentiated signal.

The particles are preferably functionalized fibrils which broadly havethe formula ##STR1## where n is an integer, L is a number less than0.1n, m is a number less than 0.5n,

each of R is the same and is selected from SO₃ H, COOH, NH₂, OH, CHO,CN, COCl, halide, COSH, SH, COOR', SR', SiR'₃, ##STR2## R", Li, AlR'₂,Hg--X, TlZ₂ and Mg--X, y is an integer equal to or less than 3,

R' is alkyl, aryl, cycloalkyl or aralkyl,

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl, fluoroaralkyl orcycloaryl,

X is halide, and

Z is carboxylate or trifluoroacetate.

The carbon atoms, C_(n), are surface carbons of a substantiallycylindrical, graphitic nanotube of substantially constant diameter. Thenanotubes include those having a length to diameter ratio of greaterthan 5 and a diameter of less than 0.5μ, preferably less than 0.1μ. Thenanotubes can also be substantially cylindrical, graphitic nanotubeswhich are substantially free of pyrolytically deposited carbon, morepreferably those characterized by having a projection of the graphitelayers on the fibril axis which extends for a distance of at least twofibril diameters and/or those having cylindrical graphitic sheets whosec-axes are substantially perpendicular to their cylindrical axis. Thesecompositions are uniform in that each of R is the same.

The particles also include non-uniformly substituted nanotubes. Theseinclude compositions of the formula ##STR3## where n, L, m, R and thenanotube itself are as defined above, provided that each of R does notcontain oxygen, or, if each of R is an oxygen-containing group COOH isnot present.

Functionalized nanotubes having the formula ##STR4## where n, L, m, Rand R' have the same meaning as above and the carbon atoms are surfacecarbon atoms of a fishbone fibril having a length to diameter ratiogreater than 5, are also included as particles within the invention.These may be uniformly or non-uniformly substituted. Preferably, thenanotubes are free of thermal overcoat and have diameters less than0.5μ.

Also included as particles in the invention are functionalized nanotubeshaving the formula ##STR5## where n, L, m, R' and R have the samemeaning as above. The carbon atoms, C_(n), are surface carbons of asubstantially cylindrical, graphitic nanotube of substantially constantdiameter. The nanotubes have a length to diameter ratio of greater than5 and a diameter of less than 0.5μ, preferably less than 0.1μ. Thenanotubes may be nanotubes which are substantially free of pyrolyticallydeposited carbon. More preferably, the nanotubes are those in which theprojection of the graphite layers on the fibril axes extends for adistance of at least two fibril diameters and/or those havingcylindrical graphitic sheets whose c-axes are substantiallyperpendicular to their cylindrical axis.

In both uniformly and non-uniformly substituted nanotubes, the surfaceatoms C_(n) are reacted. Most carbon atoms in the surface layer of agraphitic fibril, as in graphite, are basal plane carbons. Basal planecarbons are relatively inert to chemical attack. At defect sites, where,for example, the graphitic plane fails to extend fully around thefibril, there are carbon atoms analogous to the edge carbon atoms of agraphite plane (See Urry, Elementary Equilibrium Chemistry of Carbon,Wiley, New York 1989.) for a discussion of edge and basal planecarbons).

At defect sites, edge or basal plane carbons of lower, interior layersof the nanotube may be exposed. The term surface carbon includes all thecarbons, basal plane and edge, of the outermost layer of the nanotube,as well as carbons, both basal plane and/or edge, of lower layers thatmay be exposed at defect sites of the outermost layer. The edge carbonsare reactive and must contain some heteroatom or group to satisfy carbonvalency.

The substituted nanotubes described above may advantageously be furtherfunctionalized. Such compositions include compositions of the formula##STR6## where the carbons are surface carbons of a nanotube, n, L and mare as described above,

A is selected from ##STR7## --CR'₂ --OY, N═Y or C═Y, Y is an appropriatefunctional group of a protein, a peptide, an enzyme, an antibody, anucleotide, an oligonucleotide, an antigen, or an enzyme substrate,enzyme inhibitor or the transition state analog of an enzyme substrateor is selected from R'--OH, R'--NH₂, R'SH, R'CHO, R'CN, R'X, R'SiR'₃,##STR8## and w is an integer greater than one and less than 200.

The carbon atoms, C_(n), are surface carbons of a substantiallycylindrical, graphitic nanotube of substantially constant diameter. Thenanotubes include those having a length to diameter ratio of greaterthan 5 and a diameter of less than 0.1μ, preferably less than 0.05μ. Thenanotubes can also be substantially cylindrical, graphitic nanotubeswhich are substantially free of pyrolytically deposited carbon. Morepreferably they are characterized by having a projection of the graphitelayers on the fibril axes which extends for a distance of at least twofibril diameters and/or they are comprised of cylindrical graphiticsheets whose c-axes are substantially perpendicular to their cylindricalaxes. Preferably, the nanotubes are free of thermal overcoat and havediameters less than 0.5μ.

The functional nanotubes of structure ##STR9## may also befunctionalized to produce compositions having the formula ##STR10##where n, L, m, R' and A are as defined above. The carbon atoms, C_(n),are surface carbons of a substantially cylindrical, graphitic nanotubeof substantially constant diameter. The nanotubes include those having alength to diameter ratio of greater than 5 and a diameter of less than0.5μ, preferably less than 0.1μ. The nanotubes can also be substantiallycylindrical, graphitic nanotubes which are substantially free ofpyrolytically deposited carbon. More preferably they are characterizedby having a projection of the graphite layers on the fibril axes whichextends for a distance of at least two fibril diameters and/or by havingcylindrical graphitic sheets whose c-axes are substantiallyperpendicular to their cylindrical axis. Preferably, the nanotubes arefree of thermal overcoat and have diameters less than 0.5μ.

The particles of the invention also include nanotubes upon which certaincyclic compounds are adsorbed. These include compositions of matter ofthe formula ##STR11## where n is an integer, L is a number less than0.1n, m is less than 0.5n, a is zero or a number less than 10, X is apolynuclear aromatic, polyheteronuclear aromatic ormetallopolyheteronuclear aromatic moiety and R is as recited above. Thecarbon atoms, C_(n), are surface carbons of a substantially cylindrical,graphitic nanotube of substantially constant diameter. The nanotubesinclude those having a length to diameter ratio of greater than 5 and adiameter of less than 0.5μ, preferably less than 0.1μ. The nanotubes canalso be substantially cylindrical, graphitic nanotubes which aresubstantially free of pyrolytically deposited carbon and more preferablythose characterized by having a projection of the graphite layers onsaid fibril axes which extend for a distance of at least two fibrildiameters and/or those having cylindrical graphitic sheets whose c-axesare substantially perpendicular to their cylindrical axes. Preferably,the nanotubes are free of thermal overcoat and have diameters less than0.5μ.

Preferred cyclic compounds are planar macrocycles as described on p. 76of Cotton and Wilkinson, Advanced Organic Chemistry. More preferredcyclic compounds for adsorption are porphyrins and phthalocyanines.

The adsorbed cyclic compounds may be functionalized. Such compositionsinclude compounds of the formula ##STR12## where m, n, L, a, X and A areas defined above and the carbons are surface carbons of a substantiallycylindrical graphitic nanotube as described above.

The carbon fibrils functionalized as described above may be incorporatedin a matrix. Preferably, the matrix is an organic polymer (e.g., athermoset resin such as epoxy, bismaleimide, polyamide, or polyesterresin; a thermoplastic resin; a reaction injection molded resin; or anelastomer such as natural rubber, styrene-butadiene rubber, orcis-1,4-polybutadiene); an inorganic polymer (e.g., a polymericinorganic oxide such as glass), a metal (e.g., lead or copper), or aceramic material (e.g., Portland cement).

Without being bound to a particular theory, the functionalized fibrilsare better dispersed into polymer systems because the modified surfaceproperties are more compatible with the polymer, or, because themodified functional groups (particularly hydroxyl or amine groups) arebonded directly to the polymer as terminal groups. In this way, polymersystems such as polycarbonates, polyurethanes, polyesters orpolyamides/imides bond directly to the fibrils making the fibrils easierto disperse with improved adherence.

Functional groups are introduced onto the surface of carbon fibrils bycontacting carbon fibrils with a strong oxidizing agent for a period oftime sufficient to oxidize the surface of said fibrils and furthercontacting said fibrils with a reactant suitable for adding a functionalgroup to the oxidized surface. Preferably, the oxidizing agent iscomprised of a solution of an alkali metal chlorate in a strong acid. Inother embodiments the alkali metal chlorate is sodium chlorate orpotassium chlorate. In preferred embodiments the strong acid used issulfuric acid. Periods of time sufficient for oxidation are from about0.5 hours to about 24 hours.

A network of carbon fibrils are produced by contacting carbon fibrilswith an oxidizing agent for a period of time sufficient to oxidize thesurface of the carbon fibrils, contacting the surface-oxidized carbonfibrils with reactant suitable for adding a functional group to thesurface of the carbon fibrils, and further contacting thesurface-functionalized fibrils with a cross-linking agent effective forproducing a network of carbon fibrils. A preferred cross-linking agentis a polyol, polyamine or polycarboxylic acid.

The functionalized fibrils may also be in the form of rigid networks offibrils. A well-dispersed, three-dimensional network ofacid-functionalized fibrils may, for example, be stabilized bycross-linking the acid groups (inter-fibril) with polyols or polyaminesto form a rigid network.

The fibril particles also include three-dimensional networks formed bylinking functionalized fibrils of the invention. These complexes includeat least two functionalized fibrils linked by one or more linkerscomprising a direct bond or chemical moiety. These networks compriseporous media of remarkably uniform equivalent pore size.

Although the interstices between these fibrils are irregular in bothsize and shape, they can be thought of as pores and characterized by themethods used to characterize porous media. The size of the intersticesin such networks can be controlled by the concentration and level ofdispersion of fibrils, and the concentration and chain lengths of thecross-linking agents.

The complex including the particles may be collected on, e.g., anelectrode surface where it is excited and induced toelectrochemiluminescence by impressing a voltage on the electrode. Whilethe invention is preferably carried out by collecting the complex in ameasurement zone, i.e., on a surface at which it can be caused toluminesce, the invention also embraces methods wherein the complex iscollected in a measurement zone and thereafter means are brought to thatzone or other steps taken to induce and measure luminescence.

The collection of the complex may be carried out by several differentmethods, including gravity settling, filtration, centrifugation andmagnetic attraction of magnetically responsive particles which form partof the complex. The several embodiments are described in further detailbelow.

The invention is also in a method for performing a binding assay for ananalyte of interest present in a sample comprising the steps of:

(a) forming a composition containing

(i) said sample

(ii) an assay-performance-substance which contains a component linked toa label compound capable of being induced to luminesce, and

(iii) a plurality of functionalized graphitic nanotubes bound to anassay-performance-substance;

(b) incubating said composition to form a complex which includes saidfunctionalized graphitic nanotube and said label compound;

(c) collecting said complex in a measurement zone;

(d) inducing the label compound in said complex to luminesce by surfaceselective excitation, and

(e) measuring the emitted luminescence to measure the presence of theanalyte of interest in the sample.

In particular, said complex is collected on the surface of means forinducing luminescence and measuring said luminescence.

The method is advantageously based upon measurement ofelectrochemiluminescence wherein said complex is collected at anelectrode surface.

The invention is also in a method for performing a binding assay for ananalyte of interest present in a sample based upon measurement ofelectrochemiluminescence at an electrode surface comprising the steps:

(a) forming a composition containing

(i) said sample

(ii) an assay-performance-substance which contains a component linked toa label compound capable of being induced to electrochemiluminescence,and

(iii) a plurality of functionalized graphitic nanotubes bound to anassay-performance-substance;

(b) incubating said composition to form a complex which includes saidfunctionalized graphitic nanotube and said label compound;

(c) collecting said complex;

(d) causing said collected complex to come in contact with an electrodesurface and inducing the label compound in said complex to luminesce byimpressing a voltage on said electrode; and

(e) measuring the emitted luminescence at the electrode surface tomeasure the presence of the analyte of interest in the sample.

The invention is further in a method for performing a binding assay foran analyte of interest present in a sample based upon measurement ofelectrochemiluminescence at an electrode surface comprising the steps:

(a) forming a composition containing

(i) said sample

(ii) an assay-performance-substance which contains a component linked toa label compound capable of being induced to electrochemiluminesce, and

(iii) a plurality of magnetically responsive, suspended, functionalized,graphitic nanotubes bound to an assay-performance-substance;

(b) incubating said composition to form a complex which includes agraphitic nanotube and said label compound;

(c) collecting said complex by imposition of a magnetic field on saidgraphitic nanotubes;

(d) causing said collected complex to come in contact with an electrodesurface and inducing the label compound in said complex to luminescenceby imposing a voltage on said electrode; and

(e) measuring the emitted luminescence at the electrode surface tomeasure the presence of the analyte of interest in the sample.

The imposition of said magnetic field causes said complex to collect atthe surface of said electrode.

In addition, the invention is in a composition of matter for use as areagent in a microparticulate-based binding assay comprisingfunctionalized graphitic nanotubes and at least one other componentselected from the group consisting of:

(a) electrolyte;

(b) label compound containing an ECL moiety;

(c) analyte of interest or an analog of the analyte of interest;

(d) a binding partner of the analyte of interest or of its analog;

(e) a reactive component capable of reacting with (c) or (d);

(f) a reductant; and

(g) an electrochemiluminescent-reaction enhancer, provided; however,that no two components contained within any reagent composition arereactive with one another during storage so as to impair their functionin the intended assay.

The reagent may contain magnetically responsive graphitic nanotubes.

The invention is also in an assay reagent for an assay based upon abinding reaction and the measurement of an electrochemiluminescentphenomenon comprising:

(a) an electrolyte;

(b) a plurality of magnetically responsive functionalized graphiticnanotubes having a surface bound to an assay-performance-substance; and

(c) a label substance having binding properties, said label substanceincluding a chemical moiety having electrochemiluminescent properties.

The invention is further in a method for performing an assay for ananalyte of interest present in a sample comprising the steps of:

(a) forming a composition containing

(i) said sample, and

(ii) a functionalized graphitic nanotube linked to an assay componentlinked to a label compound capable of being induced to luminesce,wherein said component is a substrate of the analyte of interest;

(b) incubating said composition under conditions to permit said analyteto cleave said component;

(c) separating said functionalized graphitic nanotube from saidcomposition;

(d) inducing the label compound to luminesce; and

(e) measuring the emitted luminescence to measure the presence of theanalyte of interest in the sample.

While batch assays can be performed, continuous or semi-continuousassays can be performed in flow cells. In a flow cell, the solid-phaseremains in the measurement cell while the solution flows through andexits the cell. If the solid-phase (e.g., particles) are more dense thanwater, i.e., have a density greater than that of water, (more than 1.0g/mL) the force of gravity upon the particles causes them to fall to thebottom of the cell. The cell can be constructed such that the particlessettle to the bottom as the fluid flows through the cell or the cell canbe constructed such that the majority of the sample is contained in thecell in a columnar compartment above the working electrode of an ECLsystem. Sufficient dwell time in the cell must be provided to permit theparticles to settle on the surface of the electrode before inducing ECL.

In another embodiment of the invention, the assay composition containingsuspended fibrils having a density greater than the balance of the assaycomposition may be subjected to centrifugation in order to remove theparticles to a measurement zone where they are subsequently brought intocontact with, e.g., an electrode to induce electrochemiluminescence orbrought directly into contact with an electrode in the centrifugationstep.

In this embodiment, the measurement cell is provided with means torapidly rotate the sample and sample enclosure. Centrifugal force causesthe fibrils in the sample to move outward from the axis of rotation ofthe sample enclosure and to collect on the outer surface of the sampleenclosure. The outer surfaces of such sample enclosure may constitutethe working electrode of an ECL measurement system.

In a third embodiment, the fibrils may be removed by filtration from theassay composition. In this embodiment the particles need not have adensity greater than the balance of the assay composition. The fibrilsare separated from the solution and concentrated by drawing the solutionthrough a filter, e.g. pumping and collecting the particles on thesurface of the filter. This surface of the filter is, for example,coated with a thin metal film which can serve as the working electrodein an ECL detection system.

In another embodiment, the suspended fibrils are magneticallyresponsive, e.g. they may be paramagnetic or ferromagnetic, and arecollected in a measurement zone or, preferably, directly at the surfaceof an electrode, by imposition of a magnetic field on the particles. Themeasurement cell is equipped with a magnet. The magnetic field of themagnet applies a force on the particles as they reside in a batch cellor as they flow through a flow cell, causing them to separate from thebulk of the solution onto the surface of the cell which is in closestproximity to the magnet. If the magnet is placed in a proper orientationand in close proximity to the working electrode of an ECL detectionsystem the particles will concentrate on the surface of the workingelectrode.

Several different heterogeneous and homogeneous formats for bindingassays can be implemented using the methods described above to collectand concentrate the complex on the surface of an electrode. In aheterogeneous binding assay the complex is separated from thecomposition before measuring luminescence from the label. In homogeneousassays, no separation of the bound (to the solid phase) and unboundlabeled reagents is made.

In a homogeneous assay, when the complex is concentrated on the surfaceof the working electrode, the measured signal from the label is muchgreater than it would be in the absence of a collection step. The signalfrom the uncomplexed labeled reagents, in contrast, is not changed.Hence, despite the presence of the uncomplexed labeled reagents in themeasurement cell, the signal from the collected complex is stronger thanin an assay without collection of complex. The detection limit for thebinding assay is, much improved as a result of the collection procedure.

In a preferred embodiment of the invention, an in-situ separation stepis included in the homogeneous binding assay procedure. After the assaycomposition, i.e., sample, assay performance substance and particleshave been pumped into the measurement cell and the complex captured uponthe working electrode, a second fluid is pumped through the cell whichis free of label or labeled reagents, thereby performing an in-situ washor separation of the complex from unbound components of the assaycomposition. This assay procedure is technically a heterogeneous bindingassay. However, the ability to perform the separation inside themeasurement cell is advantageous in that it does not require additionalseparation apparatus and the procedure is generally much faster thanexternal separation methods.

Heterogeneous binding assays are conducted using the invention by mixingthe components of the assay composition and allowing them to react for apredetermined length of time. The assay composition is then subjected toa separation step wherein the solution is separated from the particles.Electrochemiluminescence is then measured from either the complex or thesolution. Measuring the ECL from the complex after a concentration steppermits measurement of analyte with better accuracy and with a lowerdetection limit than is possible without concentration.

REPRESENTATIVE EXAMPLE

In one representative embodiment of the invention the functionalizedfibril is further reacted with avidin (an assay-performance-substance)to prepare it for use in an ECL assay. The so-modified fibril is thenreacted with a biotinylated oligonucleotide (anassay-performance-substance) to form afibril-avidin-biotin-oligonucleotide complex for use in a DNA probeassay. This complex can be used as a probe for an oligonucleotideanalyte of interest which is complementary to said probe in a sandwichassay in the presence of a second oligonucleotide complementary to saidanalyte of interest labeled with an ECL moiety.

DETAILED DESCRIPTION OF THE INVENTION

The invention, as well as other objects and features thereof, will beunderstood more clearly and fully from the following description ofcertain preferred embodiments.

The invention is broadly applicable to analytes of interest which arecapable of entering into binding reactions. These reactions include,e.g., antigen-antibody, ligand receptor, DNA and RNA interactions, andother known reactions. The invention relates to different methods andassays for qualitatively and quantitatively detecting the presence ofsuch analytes of interest in a multicomponent sample.

The Samples

The sample which may contain the analyte of interest, which may be insolid, emulsion, suspension, liquid, or gas form, may be derived from,for example, cells and cell-derived products, water, food, blood, serum,hair, sweat, urine, feces, tissue, saliva, oils, organic solvents orair. The sample may further comprise, for example, water, acetonitrile,dimethyl sulfoxide, dimethyl formamide, n-methyl-pyrrolidone or alcoholsor mixtures thereof.

The Analytes

Typical analytes of interest are a whole cell or surface antigen,subcellular particle, virus, prion, viroid, antibody, antigen, hapten,fatty acid, nucleic acid, protein, lipoprotein, polysaccharide,lipopolysaccharide, glycoprotein, peptide, polypeptide, cellularmetabolite, hormone, pharmacological agent, synthetic organic molecule,organometallic molecule, tranquilizer, barbiturate, alkaloid, steroid,vitamin, amino acid, sugar, lectin, recombinant or derived protein,biotin, avidin, streptavidin, or inorganic molecule present in thesample. Typically, the analyte of interest is present at a concentrationof 10⁻³ molar or less, for example, as low as 10⁻¹² molar or lower.

Assay-Performance-Substance

The assay-performance-substance which is combined with the samplecontaining the analyte of interest contains at least one substanceselected from the group consisting of (i) added analyte of interest orits analog, as defined above, (ii) a binding partner of the analyte ofinterest or its said analog, and (iii) a reactive component, as definedabove, capable of binding with (i) or (ii), wherein one of saidsubstances is linked to a compound or moiety, e.g. an ECL moiety capableof being induced to luminesce. The labeled substance may be a whole cellor surface antigen, a subcellular particle, virus, prion, viroid,antibody, antigen, hapten, lipid, fatty acid, nucleic acid,polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein,peptide, polypeptide, cellular metabolite, hormone, pharmacologicalagent, tranquilizer, barbiturate, alkaloid, steroid, vitamin, aminoacid, sugar, nonbiological polymer (preferably soluble), lectin,recombinant or derived protein, synthetic organic molecule,organometallic molecule, inorganic molecule, biotin, avidin orstreptavidin. In one embodiment, the reagent is anelectrochemiluminescent moiety conjugated to an antibody, antigen,nucleic acid, hapten, small nucleotide sequence, oligomer, ligand,enzyme, or biotin, avidin, streptavidin, Protein A, Protein G, orcomplexes thereof, or other secondary binding partner capable of bindingto a primary binding partner through protein interactions.

Analogs of the analyte of interest, which can be natural or synthetic,are typically compounds which have binding properties comparable to theanalyte, but can also be compounds of higher or lower bindingcapability. The reactive components capable of binding with the analyteor its analog, and/or with a binding partner thereof, and through whichthe ECL moiety can be linked to the analyte, is suitably a secondantibody or a protein such as Protein A or Protein G, or avidin orbiotin or another component known in the art to enter into bindingreactions.

The Labels

Advantageously, the ECL moieties are metal chelates. The metal of thatchelate is suitably any metal such that the metal chelate will luminesceunder the electrochemical conditions which are imposed on the reactionsystem in question. The metal of such metal chelates is, for instance, atransition metal (such as a d-block transition metal) or a rare earthmetal. The metal is preferably ruthenium, osmium, rhenium, iridium,rhodium, platinum, indium, palladium, molybdenum, technetium, copper,chromium or tungsten. Especially preferred are ruthenium and osmium.

The ligands which are linked to the metal in such chelates are usuallyheterocyclic or organic in nature, and play a role in determiningwhether or not the metal chelate is soluble in an aqueous environment orin an organic or other nonaqueous environment. The ligands can bepolydentate, and can be substituted. Polydentate ligands includearomatic and aliphatic ligands. Suitable aromatic polydentate ligandsinclude aromatic heterocyclic ligands. Preferred aromatic heterocyclicligands are nitrogen-containing, such as, for example, bipyridyl,bipyrazyl, terpyridyl, and phenanthrolyl. Suitable substituents includefor example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl,substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano,amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine,guanidinium, ureide, sulfur-containing groups, phosphorus containinggroups, and the carboxylate ester of N-hydroxysuccinimide. The chelatemay have one or more monodentate ligands, a wide variety of which areknown to the art. Suitable monodentate ligands include, for example,carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromaticand heterocyclic phosphines, amines, stilbenes, and arsines.

Examples of suitable chelates are bis(4,4'-carbomethoxy)-2,2'-bipyridine! 2-3-(4-methyl-2,2'-bipyridine-4-yl)propyl!-1,3-dioxolane ruthenium (II);bis (2,2'bipyridine) 4-(butan-1-al)-4'-methyl-2,2'-bipyridine! ruthenium(II); bis (2,2'-bipyridine) 4-(4'-methyl-2,2'-bipyridine-4'-yl)-butyricacid! ruthenium (II); tris (2,2'bipyridine) ruthenium (II);(2,2'-bipyridine) bis-bis(1,2-diphenylphosphino)ethylene! 23-(4-methyl-2,2'-bipyridine-4'-yl)propyl!-1,3-dioxolane osmium (II); bis(2,2'-bipyridine) 4-(4'-methyl-2,2'-bipyridine)-butylamine! ruthenium(II); bis (2,2'-bipyridine)1-bromo-4(4'-methyl-2,2'-bipyridine-4-yl)butane! ruthenium (II); bis(2,2'-bipyridine)maleimidohexanoic acid,4-methyl-2,2'-bipyridine-4'-butylamide ruthenium (II). Other ECLmoieties are described in commonly assigned PCT published applicationUS87/00987 and PCT published application 88/0394.

The function of the ECL moieties is to emit electromagnetic radiation asa result of introduction into the reaction system of electrochemicalenergy. In order to do this, they must be capable of being stimulated toan excited energy state and also capable of emitting electromagneticradiation, such as a photon of light, upon descending from that excitedstate. While not wishing to be bound by theoretical analysis of themechanism of the ECL moiety's participation in theelectrochemiluminescent reaction, we believe that it is oxidized by theintroduction of electrochemical energy into the reaction system andthen, through interaction with a reductant present in the system, isconverted to the excited state. This state is relatively unstable, andthe metal chelate quickly descends to a more stable state. In so doing,the chelate gives off electromagnetic radiation, such as a photon oflight, which is detectable.

The amount of metal chelate or other metal-containing ECL moietyincorporated in accordance with the invention will vary from system tosystem. Generally, the amount of such moiety utilized is that amountwhich is effective to result in the emission of a detectable, and ifdesired, quantitatable, emission of electromagnetic energy, from theaforementioned composition or system. The detection and/or quantitationof an analyte of interest is typically made from a comparison of theluminescence from a sample containing an analyte of interest and an ECLmoiety to the luminescence emitted by a calibration standard developedwith known amounts of the analyte of interest and ECL moiety. Thisassumes a homogeneous format. In the heterogeneous mode, a separation asdiscussed previously is carried out prior to ECL analysis.

As can be appreciated by one of ordinary skill in the art, the identityand amount of the metal-containing ECL moiety will vary from one systemto another, depending upon prevailing conditions. The appropriatemetal-containing ECL moiety, and sufficient amount thereof to obtain thedesired result, can be determined empirically by those of ordinary skillin the art, once equipped with the teachings herein, without undueexperimentation.

Graphitic Nanotubes

The nanotubes may be used as a solid support for analytical applicationsin various geometries including as dispersed, as aggregates, as mats orfilms, attached to larger supports including beads, or mixed withanother material and used as a composite, for example in a porouscolumn. Nanotubes primarily consist of chemically-modifiable graphiticcarbon. They generally have diameters no greater than 0.1 μm and lengthto diameter ratios of at least 5. Typically, they have diameters of 0.01μm and lengths of 1-10 μm.

Functionalized Nanotubes

Advantageously, the fibrils are functionalized fibrils, i.e. fibrilswhose surfaces are uniformly or non-uniformly modified so as to have afunctional chemical moiety associated therewith. The fibril surfaces maybe functionalized by reaction with oxidizing or other chemical media.The fibril surfaces may be uniformly modified either by chemicalreaction or by physical adsorption of species which themselves have achemical reactivity. The fibril surfaces may be modified e.g. byoxidation and may be further modified by reaction with other functionalgroups. The fibril surfaces may be modified with a spectrum offunctional groups so that the fibrils can be chemically reacted orphysically bonded to chemical groups in a variety of substratesincluding binding components in electrochemiluminescence assays.

Complex structures of fibrils may be obtained by linking functionalgroups on the fibrils with one another by a range of linker chemistries.

Methods for chemical modification of fibril surfaces and methods forphysically adsorbing species on the surfaces of fibrils are described soas to provide, in each case, a functional moiety associated with thesurface of the fibril.

Functionalized Nanotubes Bound to Assay-Performance-Substances

The functionalized fibrils may be reacted with various biomolecules toprepare them for use in ECL assays. Variousassay-performance-substances, e.g. antibodies, receptors, or otherbinding moieties, can be reacted with the functionalized fibrils toprepare them for use in ECL assays.

The Particles

Graphitic nanotubes may be covalently or noncovalently attached tolarger solid phases, such as particles.

The particles advantageously comprise micro-particulate matter having adiameter of 0.05 μm to 200 μm, preferably 0.1 μm to 100 μm, mostpreferably 0.5 μm to 10 μm, and a surface component capable of bindingto the analyte and/or one or more of the other substances defined insubparagraphs (b)(i), (b)(ii), or (b)(iii) above. For example, themicroparticulate matter may be crosslinked starch, dextrans, cellulose,proteins, organic polymers, styrene copolymer such as styrene/butadienecopolymer, acrylonitrile/butadiene/styrene copolymer, vinylacetylacrylate copolymer, or vinyl chloride/acrylate copolymer, inertinorganic particles, chromium dioxide, oxides of iron, silica, silicamixtures, and proteinaceous matter, or mixtures thereof. Desirably, theparticles are suspended in the ECL system.

Assay Media

In order to operate a system in which an electrode introduceselectrochemical energy, it is necessary to provide an electrolyte inwhich the electrode is immersed and which contains the ECL moiety. Theelectrolyte is a phase through which charge is carried by ions.Generally, the electrolyte is in the liquid phase, and is a solution ofone or more salts or other species in water, an organic liquid ormixture of organic liquids, or a mixture of water and one or moreorganic liquids. However, other forms of electrolyte are also useful incertain embodiments of the invention. For example, the electrolyte maybe a dispersion of one or more substances in a fluid--e.g., a liquid, avapor, or a supercritical fluid--or may be a solution of one or moresubstances in a solid, a vapor or supercritical fluid.

The electrolyte is suitably a solution of a salt in water. The salt canbe a sodium salt or a potassium salt preferably, but incorporation ofother cations is also suitable in certain embodiments, as long as thecation does not interfere with the electrochemiluminescent interactionsequence. The salt's anion may be a phosphate, for example, but the useof other anions is also permissible in certain embodiments of theinvention once--again, as long as the selected anion does not interferewith the electrochemiluminescent interaction sequence.

The composition may also be nonaqueous. While supercritical fluids canin certain instances be employed advantageously, it is more typical toutilize an electrolyte comprising an organic liquid in a nonaqueouscomposition. Like an aqueous electrolyte, the nonaqueous electrolyte isalso a phase through which charge is carried by ions. Normally, thismeans that a salt is dissolved in the organic liquid medium. Examples ofsuitable organic liquids are acetonitrile, dimethylsulfoxide (DMSO),dimethylformamide (DMF), methanol, ethanol, and mixtures of two or moreof the foregoing. Illustratively, tetraalkylammonium salts, such astetrabutylammonium tetrafluoroborate, which are soluble in organicliquids can be used with them to form nonaqueous electrolytes.

The electrolyte is, in certain embodiments of the invention, a bufferedsystem. Phosphate buffers are often advantageous. Examples are anaqueous solution of sodium phosphate/sodium chloride, and an aqueoussolution of sodium phosphate/sodium fluoride.

Other Assay Components

As described PCT published application U.S. 89/04859 commonly assigned,entitled Electrochemiluminescent Reaction Utilizing Amine-DerivedReductant, the disclosure of which is incorporated by reference, it isdesirable to include a reductant, typically an amine or amine moiety (ofa larger molecule) which can be oxidized and spontaneously decomposed toconvert it into a highly reducing species. It is believed that the amineor amine moiety is also oxidized by electrochemical energy introducedinto the reaction system. The amine or amine moiety loses one electron,and then deprotonates, or rearranges itself, into a strong reducingagent. This agent interacts with the oxidized metal-containing ECLmoiety and causes it to assume the excited state discussed above. Inorder to carry out its role, the amine or amine moiety preferably has acarbon-centered radical with an electron which can be donated from suchcarbon, and an alpha carbon which can then act as a proton donor duringdeprotonation in order to form the reductant. The amine-derivedreductant provides the necessary stimulus for converting themetal-containing ECL moiety to its excited state, from which detectableelectromagnetic radiation is emitted.

A wide range of amines and corresponding amine moieties can be utilizedin practicing the present invention. Generally, the amine or aminemoiety is chosen to suit the pH of the system which is to beelectrochemiluminescently analyzed. Another relevant factor is that theamine or amine moiety should be compatible with the environment in whichit must function during analysis, i.e., compatible with an aqueous ornonaqueous environment, as the case may be. Yet another consideration isthat the amine or amine moiety selected should form an amine-derivedreductant under prevailing conditions which is strong enough to reducethe oxidized metal-containing ECL moiety in the system.

Amines (and corresponding moieties derived therefrom) which areadvantageously utilized in the present invention are aliphatic amines,such as primary, secondary and tertiary alkyl amines, the alkyl groupsof each having from one to three carbon atoms, as well as substitutedaliphatic amines. Tripropyl amine is an especially preferred amine as itleads to, comparatively speaking, a particularly high-intensity emissionof electromagnetic radiation, which enhances the sensitivity andaccuracy of detection and quantitation with embodiments in which it isused. Also suitable are diamines, such as hydrazine, and polymines, suchas poly(ethyleneimine). Examples of other amines suitable for practicingthe invention are triethanol amine, triethyl amine,1,4-diazabicyclo-(2.2.2)-octane, 1-piperidine ethanol,1,4-piperazine-bis-(ethane-sulfonic acid), tri-ispropyl amine andpoly(ethyleneimine).

Typically, the metal-containing ECL moiety utilized in the presentinvention is the reaction-limiting constituent. Accordingly, it is alsotypical that the amine or amine moiety is provided in a stoichiometricexcess with respect thereto. Illustratively, the amine or amine moietyis employed in a concentration of 50-150 mM. For utilization at a pH ofapproximately 7, a concentration of 100 mM is often advantageous. Incertain embodiments, the upper limit on amine or amine moietyconcentration is determined by the maximum solubility of the amine ormoiety in the environment in which it is being used, for example inwater. In general, the amount of amine or amine moiety employed is thatwhich is sufficient to effect the transformation of the oxidizedmetal-containing ECL moiety into its excited state so that luminescenceoccurs. Those of ordinary skill in the art, equipped with the teachingsherein, can determine empirically the amount of amine or amine moietyadvantageously used for the particular system being analyzed, withoutundue experimentation.

As described in commonly assigned PCT published application US 89/04915,entitled Enhanced Electrochemiluminescent Reaction, the contents ofwhich are incorporated by reference, the assays of the invention aredesirably carried out in the presence of an enhancer, typically acompound of the formula ##STR13## wherein R is hydrogen or C_(n)H_(n2+1), R' is C_(n) H_(2n), x is 0 to 70, and n is from 1 to 20.Specifically, n is from 1 to 4. Specific examples are a substanceavailable in commerce under the name Triton X-100, of the formula##STR14## wherein x is 9-10, and a substance available in commerce underthe name Triton N-401 (NPE-40), of the formula ##STR15## wherein x is40. The enhancer is generally utilized in an amount sufficient so thatin its presence the desired increase in emission of electromagneticradiation occurs. Typically, the amount is 0.01% to 5.0%, morespecifically 0.1% to 1.0%, v/v. The subject matter of this applicationis incorporated by reference.

The ECL moiety incorporated in accordance with the present invention isinduced to emit electromagnetic radiation by stimulating it into anexcited state. This is accomplished by exposing the system in which theECL moiety is incorporated to electrochemical energy. The potential atwhich oxidation of the ECL moiety and the species forming a strongreductant occurs depends upon the exact chemical structures thereof, aswell as factors such as the pH of the system and the nature of theelectrode used to introduce electrochemical energy. It is well known tothose of ordinary skill in the art how to determine the optimalpotential and emission wavelength of an electrochemiluminescent system.Certain preferred methods of stimulating the ECL system are disclosed incommonly assigned PCT published application US 89/01814, the contents ofwhich are incorporated herein by reference.

Apparatus for Measuring Electrochemiluminescence

An apparatus for carrying out the assays of the invention is describedin FIGS. 1 and 2. FIG. 1 discloses an advantageous ECL apparatus, butthe methods of the present invention are not limited to application inapparatus 10, but rather may be employed in other types of ECL apparatuswhich include a working electrode or other triggering surface to provideelectrochemical energy to trigger the ECL moiety intoelectrochemiluminescence. While the methods of the invention can becarried out in a static or flow-through mode, apparatus 10 includes aflow-through cell, which provides distinct advantages for many types ofsamples including binding-assay samples. Further details of apparatusfor carrying out the ECL assays of the invention are disclosed incommonly assigned published PCT applications US 89/04854 and U.S.90/01370.

Apparatus 10 includes an electrochemical cell 12, a lightdetection/measurement device 14, which may advantageously be aphotomultiplier tube (PMT), photodiode, charge coupled device,photographic film or emulsion or the like, and a pump 16, which isadvantageously a peristaltic pump, to provide for fluid transport to,through and from cell 12. Alternatively, a positive displacement pumpmay be used. A shutter mechanism 18 is provided between cell 12 and PMT14 and is controllably operated to open only so far as to expose PMT 14to cell 12 during ECL measurement periods. The shutter mechanism may beclosed, for example, during maintenance. Also included in apparatus 10but not illustrated in FIG. 1 is a lightproof housing intended to mountthe various components therein and to shield PMT 14 from any externallight during the ECL measurements.

Cell 12 itself includes a first mounting block 20 through which passesan inlet tube 22 and an outlet tube 24, which may be advantageouslyconstructed of stainless steel. Mounting block 20 has a first, outersurface 26 and a second, inner surface 28 defining one side of asample-holding volume 30 of cell 12 in which cell 12 holds the cleaningand/or conditioning and/or measurement solutions during correspondingoperations of apparatus 10. Inlet and outlet tubes 22, 24 pass throughmounting block 20 from outer surface 26 to inner surface 28 and openinto sample-holding volume 30. A second mounting block 32,advantageously constructed of stainless steel also has a first, outersurface 34 and a second, inner surface 36. Second mounting block 32 isseparated from first mounting block 20 by an annular spacer 38,advantageously constructed of Teflon or other non-contaminable material.Thus, outer surface 34 of mounting block 30 defines part of the secondside of the sample-holding volume 30. Spacer 38 has an outer portion 40and a central aperture 42 whose inner edge 44 defines the side wall ofsample-holding volume 30. Outer portion 40 seals the inner surface 28 offirst mounting block 20 to outer surface 34 of second mounting block 32to prevent any solution from passing out from sample-holding volume 30between the two surfaces 28, 34. Mounting block 32 further has a centralaperture 46 in which a window 48 is seal-fitted to define the rest ofthe second side of sample-holding volume 30 as a continuation of outersurface 34. Window 48 is formed of a material which is substantiallytransparent at the wavelength of electrochemiluminescent light emittedby the ECL moiety. Window 48 is therefore advantageously formed ofglass, plastic, quartz or the like.

Inlet tube 22 intersects sample-holding volume 30 at a first end 50thereof adjacent to spacer 38 and outlet tube 24 intersectssample-holding volume 30 at a second end 52 thereof, adjacent spacer 38.The combination of inlet tube 22, sample-holding volume 30 and outlettube 24 thereby provides a continuous flow path for the narrow,substantially laminar flow of a solution to, through and from cell 12.

Mounted on inner surface 28 of first mounting block 20 is a workingelectrode system 54 which, in the illustrated embodiment, includes firstand second working electrodes 56 and 58. In other embodiments, a singleworking electrode may advantageously be provided, or only electrode 56may be a working electrode. Working electrodes 56, 58 are where theelectrochemical and ECL reactions of interest can take place. Workingelectrodes 56, 58 are solid voltammetric electrodes and may therefore beadvantageously constructed of platinum, gold, carbons or other materialswhich are effective for this purpose. Wire connectors 60, 62 connectedto working electrodes 56, 58, respectively, pass out through firstmounting block 20.

Connectors 60, 62 are both connected to a first, "working electrode"terminal 64 of a voltage control 66, illustrated in FIG. 2. Voltagecontrol 66 advantageously operates in the manner of a potentiostat tosupply voltage signals to working electrodes 56, 58 and optionally tomeasure current flowing therefrom during an ECL measurement.Alternatively, connectors 60, 62 may be connected to separate terminalsof voltage control 66 for individual operation.

The potentiostat operation of voltage control 66 is further effectedthrough a counter electrode 68 and, optionally but advantageously, areference electrode 70. In the illustrated embodiment, mounting block 32is made of stainless steel and counter electrode 68 consists in exposedsurfaces 72, 74 of mounting block 32. Counter electrode 72, 74 andworking electrodes 56, 58 provide the interface to impress the potentialon the solution within sample-holding volume 30 which energizes thechemical reactions and triggers electrochemiluminescence in the sampleand/or provides energy for cleaning and conditioning the surfaces ofcell 12. Counter electrode 72, 74 is connected by a wire connector 76 toa second, "counter electrode" terminal 78 of voltage control 66.

Reference electrode 70 provides a reference voltage to which the voltageapplied by the working electrodes 56, 58 is referred, for example, +1.2volts versus the reference. Reference electrode 70 is advantageouslylocated in outlet tube 24 at a position 80 spaced from cell 12 and isconnected through a wire connector 82 to a third "reference electrode"terminal 84 of voltage control 66. In the three electrode mode, currentdoes not flow through reference electrode 70. Reference electrode 70 maybe used in a three electrode mode of operation to provide a poised,known and stable voltage and is therefore advantageously constructed ofsilver/silver chloride (Ag/AgCl) or is a saturated calomel electrode(SCE). Voltage control 66 may be operable in a two electrode mode ofoperation using only working electrode 56 and electrode 58 as acounter/reference electrode. In this two electrode mode of operation,counter/reference electrode 58 is electrically connected to voltagecontrol terminals 78 and 84 on voltage control 66. In this case, voltagecontrol 66 operates essentially as a battery. Voltage control 66supplies voltage signals to working and counter electrodes 56 and 58 andoptionally measures the current flowing through the respectiveelectrodes. Reference electrode 70 may alternatively be a so-called"quasi-reference" electrode constructed of platinum, gold, stainlesssteel or other material, which provides a less stable voltage, yet onethat is measurable with respect to the solution in contact. In both thetwo and three electrode mode, the reference electrode 70 or 58 servesthe purpose of providing a reference against which the voltage appliedto working electrodes 56 is measured. The poised voltage reference iscurrently considered to be more advantageous. Voltage control 66 in itspotentiostat operation controls the various electrodes by providing aknown voltage at working electrodes 56, 58 with respect to referenceelectrode 70 while measuring the current flow between working electrodes56, 58 and counter electrode 72, 74. Potentiostats for this purpose arewell known, and the internal structure of voltage control 66 maytherefore correspond to any of the conventional, commercially availablepotentiostats which produce the above-recited functions and so do notform a part of the present invention per se. Indeed, apparatus 10 mayalternatively be constructed without an internal voltage control 66, andmay be adapted to be connected to an external potentiostat which isseparately controlled for providing the required voltage signals toelectrodes 56, 58, 72, 74 and 70. These voltage signals, applied in aspecific manner as described below, provide repeatable initialconditions for the surfaces of working electrodes 56, 58 andadvantageously for the surfaces of cell 12 as a whole, a feature whichcontributes significantly to improved precision in ECL measurements.

Pump 16 is advantageously positioned at outlet tube 24 to "pull"solution from a sample volume in the direction of arrow A into inlettube 22. The solution will flow through inlet tube 22, sample-holdingvolume 30 and outlet tube 24 past reference electrode 70 and out in thedirection of arrow B. Alternatively, pump 16 may be positioned at inlettube 22 to "push" the solution through apparatus 10. Advantageously,this same flow path through inlet tube 22, sample-holding volume 30 andoutlet tube 24 is used for all solutions and fluids which pass throughcell 12, whereby each fluid performs a hydrodynamic cleaning action inforcing the previous fluid out of cell 12. Pump 16 may be controlled tosuspend its operation to hold a particular solution in cell 12 for anyperiod of time.

The flow-through construction of apparatus 10 permits working electrodesto be impressed with a variable voltage or to be continuously held at apreoperative potential while being continuously exposed to one or moresolutions without exposing working electrodes 56, 58 (or counter andreference electrodes 72, 74, 70 ) to air. Exposure to air, which opensthe circuit to the reference electrode 70, permits unknown, randomvoltage fluctuations which destroy the reproducibility of surfaceconditions on working electrodes 56, 58. The flow-through constructionpermits the rapid alternation between initializing steps, in whichelectrode system 54 is cleaned and conditioned, and measurement steps,in which one or more measurement waveforms or sweeps trigger ECL.

The invention is also directed to reagent compositions. Broadly, thereagent compositions may be any one of the components of the assaysystems of the invention, i.e., (a) electrolyte, (b) label compoundcontaining an ECL moiety, (c) functionalized fibrils to which anassay-performance-substance is bound, optionally bound to particles,including magnetically responsive particles, (d) analyte of interest oran analog of the analyte of interest, (e) a binding partner of theanalyte of interest or of its analog, (f) a reactive component capableof reacting with (d) or (e), (g) a reductant, or (h) anelectrochemiluminescence-reaction enhancer. The reagents may be combinedwith one another for convenience of use, i.e., two component, threecomponent, and higher multiple component mixtures may be prepared,provided that the components are not reactive with one another duringstorage so as to impair their function in the intended assay. Desirably,the reagents are two-component or multicomponent mixtures which containparticles as well as one or more other components.

The invention is also directed to kits. The kits may include vesselscontaining one or more of the components (a) to (h) recited above or thekits may contain vessels containing one or more reagent compositions asdescribed above comprising mixtures of those components, all for use inthe assay methods and systems of the invention.

METHODS OF FUNCTIONALIZING FIBRILS

The functionalized fibrils of the invention can be directly prepared bysulfonation, electrophilic addition to deoxygenated fibril surfaces ormetallation. When arc grown nanofibers are used, they may requireextensive purification prior to functionalization. Ebbesen et al.(Nature 367 519 (1994)) give a procedure for such purification.

Preferably, the carbon fibrils are processed prior to contacting themwith the functionalizing agent. Such processing may include dispersingthe fibrils in a solvent. In some instances the carbon fibrils may thenbe filtered and dried prior to further contact.

1. SULFONATION

Background techniques are described in March, J. P., Advanced OrganicChemistry, 3rd Ed. Wiley, New York 1985; House, H., Modern SyntheticReactions, 2nd Ed., Benjamin/Cummings, Menlo Park, Calif. 1972.

Activated C--H (including aromatic C--H) bonds can be sulfonated usingfuming sulfuric acid (oleum), which is a solution of conc. sulfuric acidcontaining up to 20% SO₃. The conventional method is via liquid phase atT˜80° C. using oleum; however, activated C--H bonds can also besulfonated using SO₃ in inert, aprotic solvents, or SO₃ in the vaporphase. The reaction is: p1 --C--H+SO₃ →--C--SO₃ H

Over-reaction results in formation of sulfones, according to thereaction:

2--C--H+SO₃ →--C--SO₂ --C--+H₂ O

Preparation A Activation of C--H Bonds Using Sulfuric Acid

Reactions were carried out in the gas phase and in solution without anysignificant difference in results. The vapor phase reaction was carriedout in a horizontal quartz tube reactor heated by a Lindberg furnace. Amulti-neck flask containing 20% SO₃ in conc. H₂ SO₄ fitted with gasinlet/outlet tubes was used as the SO₃ source.

A weighed sample of fibrils (BN or CC) in a porcelain boat was placed inthe 1" tube fitted with a gas inlet; the outlet was connected to a conc.H₂ SO₄ bubbler trap. Argon was flushed through the reactor for 20 min toremove all air, and the sample was heated to 300° C. for 1 hour toremove residual moisture. After drying, the temperature was adjusted toreaction temperature under argon.

When the desired temperature was stabilized, the SO₃ source wasconnected to the reactor tube and an argon stream was used to carry SO₃vapors into the quartz tube reactor. Reaction was carried out for thedesired time at the desired temperature, after which the reactor wascooled under flowing argon. The fibrils were then dried at 90° C. at 5"Hg vacuum to obtain the dry weight gain. Sulfonic acid (--SO₃ H) contentwas determined by reaction with 0.100N NaOH and back-titration with0.100N HCl using pH 6.0 as the end point.

The liquid phase reaction was carried out in conc. sulfuric acidcontaining 20% SO₃ in a multi-neck 100 cc flask fitted with athermometer/temperature controller and a magnetic stirrer. A fibrilslurry in conc. H₂ SO₄ (50) was placed in the flask. The oleum solution(20 cc) was preheated to ˜60° C. before addition to the reactor. Afterreaction, the acid slurry was poured onto cracked ice, and dilutedimmediately with 1 l DI water. The solids were filtered and washedexhaustively with DI water until there was no change in pH of the washeffluent. Fibrils were dried at 100° C. at 5" Hg vacuum. Due to transferlosses on filtration, accurate weight gains could not be obtained.Results are listed in Table I.

                                      TABLE I    __________________________________________________________________________    Summary of Reactions               SAMPLE                    FIBRIL     DRY Wt                                    SO.sub.3 H CONC    X.      RUN #           REACT               Wt. g                    TYPE                        T°C.                           TIME                               GAIN meq/g    __________________________________________________________________________    1A      118-60A           Vap 0.20 CY  110                           15 m                               9.3% 0.50    1B      118-61A           Vap 0.20 BN  100                           30 m                               8.5% 0.31    1C      118-61B           Vap 0.20 BN  65 15 m                               4.2% 0.45    1D      118-56A           Liq 1.2  CY  50 10 m     0.33    1E      118-56B           Liq 1.0  CY  25 20 m     0.40    __________________________________________________________________________

There was no significant difference in sulfonic acid content by reactionin the vapor phase or liquid phase. There was a temperature effect.Higher temperature of reaction (vapor phase) gives higher amounts ofsulfones. In 118-61B, the 4.2% wt gain agreed with the sulfonic acidcontent (theoretical was 0.51 meq/g). Runs 60A and 61A had too high a wtgain to be accounted for solely by sulfonic acid content. It wastherefore assumed that appreciable amounts of sulfones were also made.

2. ADDITIONS TO OXIDE-FREE FIBRIL SURFACES

Background techniques are described in Urry, G., Elementary EquilibriumChemistry of Carbon, Wiley, New York 1989.

The surface carbons in fibrils behave like graphite, i.e., they arearranged in hexagonal sheets containing both basal plane and edgecarbons. While basal plane carbons are relatively inert to chemicalattack, edge carbons are reactive and must contain some heteroatom orgroup to satisfy carbon valency. Fibrils also have surface defect siteswhich are basically edge carbons and contain heteroatoms or groups.

The most common heteroatoms attached to surface carbons of fibrils arehydrogen, the predominant gaseous component during manufacture; oxygen,due to its high reactivity and because traces of it are very difficultto avoid; and H₂ O, which is always present due to the catalyst.Pyrolysis at ˜1000° C. in a vacuum will deoxygenate the surface in acomplex reaction with unknown mechanism, but with known stoichiometry.The products are CO and CO₂, in a 2:1 ratio. The resulting fibrilsurface contains radicals in a C₁ -C₄ alignment which are very reactiveto activated olefins. The surface is stable in a vacuum or in thepresence of an inert gas, but retains its high reactivity until exposedto a reactive gas. Thus, fibrils can be pyrolized at ˜1000° C. in vacuumor inert atmosphere, cooled under these same conditions and reacted withan appropriate molecule at lower temperature to give a stable functionalgroup. Typical examples are: ##STR16## followed by: ##STR17## where R'is a hydrocarbon radical (alkyl, cycloalkyl, etc.)

Preparation B Preparation of Functionalized Fibrils by Reacting AcrylicAcid with Oxide-Free Fibril Surfaces

One gram of BN fibrils in a porcelain boat is placed in a horizontal 1"quartz tube fitted with a thermocouple and situated in a Lindberg tubefurnace. The ends are fitted with a gas inlet/outlets. The tube ispurged with dry, deoxygenated argon for 10 minutes, after which thetemperature of the furnace is raised to 300° C. and held for 30 minutes.Thereafter, under a continued flow of argon, the temperature is raisedin 100° C. increments to 1000° C., and held there for 16 hours. At theend of that time, the tube is cooled to room temperature (RT) underflowing argon. The flow of argon is then shunted to pass through amulti-neck flask containing neat purified acrylic acid at 50° C. andfitted with gas inlet/outlets. The flow of acrylic acid/argon vapors iscontinued at RT for 6 hours. At the end of that time, residual unreactedacrylic acid is removed, first by purging with argon, then by vacuumdrying at 100° C. at <5" vacuum. The carboxylic acid content isdetermined by reaction with excess 0.100N NaOH and back-titrating with0.100N HCl to an endpoint at pH 7.5.

Preparation C Preparation of Functionalized Fibrils by Reacting AcrylicAcid with Oxide-Free Fibril Surfaces

The procedure is repeated in a similar manner to the above procedure,except that the pyrolysis and cool-down are carried out at 10⁻⁴ Torrvacuum. Purified acrylic acid vapors are diluted with argon as in theprevious procedure.

Preparation D Preparation of Functionalized Fibrils by Reacting MaleicAcid with Oxide-Free Fibril Surfaces

The procedure is repeated as in Preparation B, except that the reactantat RT is purified maleic anhydride (MAN) which is fed to the reactor bypassing argon gas through a molten MAN bath at 80° C.

Preparation E Preparation of Functionalized Fibrils by Reacting AcryloylChloride with Oxide-Free Fibril Surfaces

The procedure is repeated as in Preparation B, except that the reactantat RT is purified acryloyl chloride, which is fed to the reactor bypassing argon over neat acryloyl chloride at 25° C. Acid chloridecontent is determined by reaction with excess 0.100N NaOH andback-titration with 0.100N HCl.

Pyrolysis of fibrils in vacuum deoxygenates the fibril surface. In a TGAapparatus, pyrolysis at 1000° C. either in vacuum or in a purified Arflow gives an average wt loss of 3% for 3 samples of BN fibrils. Gaschromatographic analyses detected only CO and CO₂, in ˜2:1 ratio,respectively. The resulting surface is very reactive and activatedolefins such as acrylic acid, acryloyl chloride, acrylamide, acrolein,maleic anhydride, allyl amine, allyl alcohol or allyl halides will reacteven at room temperature to form clean products containing only thatfunctionality bonded to the activated olefin. Thus, surfaces containingonly carboxylic acids are available by reaction with acrylic acid ormaleic anhydride; surf only acid chloride by reaction with acryloylchloride; only aldehyde from acrolein; only hydroxyl from allyl alcohol;only amine from allyl amine, and only halide from allyl halide.

3. METALLATION

Background techniques are given in March, Advanced Organic Chemistry,3rd ed., p. 545.

Aromatic C--H bonds can be metallated with a variety of organometallicreagents to produce carbon-metal bonds (C--M). M is usually Li, Be, Mg,Al, or Tl; however, other metals can also be used. The simplest reactionis by direct displacement of hydrogen in activated aromatics:

1. Fibril-H+R-Li→Fibril-Li+RH

The reaction may require additionally, a strong base, such as potassiumt-butoxide or chelating diamines. Aprotic solvents are necessary(paraffins, benzene).

2. Fibril-H+AlR₃ →Fibril-AlR₂ +RH

3. Fibril-H+Tl(TFA)₃ →Fibril-Tl(TFA)₂ +HTFA TFA=TrifluoroacetateHTFA=Trifluoroacetic acid

The metallated derivatives are examples of primary singly-functionalizedfibrils. However, they can be reacted further to give other primarysingly-functionalized fibrils. Some reactions can be carried outsequentially in the same apparatus without isolation of intermediates.##STR18##

Preparation F Preparation of Fibril-Li

One gram of CC fibrils is placed in a porcelain boat and inserted into a1" quartz tube reactor which is enclosed in a Lindberg tube furnace. Theends of the tube are fitted with gas inlet/outlets. Under continuousflow of H₂, the fibrils are heated to 700° C. for 2 hours to convert anysurface oxygenates to C--H bonds. The reactor is then cooled to RT underflowing H₂.

The hydrogenated fibrils are transferred with dry, de-oxygenated heptane(with LiAlH₄) to a 1 liter multi-neck round bottom flask equipped with apurified argon purging system to remove all air and maintain an inertatmosphere, a condenser, a magnetic stirrer and rubber septum throughwhich liquids can be added by a syringe. Under an argon atmosphere, a 2%solution containing 5 mmol butyllithium in heptane is added by syringeand the slurry stirred under gentle reflux for 4 hours. At the end ofthat time, the fibrils are separated by gravity filtration in an argonatmosphere glove box and washed several times on the filter with dry,deoxygenated heptane. Fibrils are transferred to a 50 cc r.b. flaskfitted with a stopcock and dried under 10⁻⁴ torr vacuum at 50° C. Thelithium concentration is determined by reaction of a sample of fibrilswith excess 0.100N HCl in DI water and back-titration with 0.100N NaOHto an endpoint at pH 5.0.

Preparation G Preparation of Fibril-Tl(TFA)₂

One gram of CC fibrils are hydrogenated as in Preparation E and loadedinto the multi-neck flask with HTFA which has been degassed by repeatedpurging with dry argon. A 5% solution of 5 mmol Tl(TFA)₃ in HTFA isadded to the flask through the rubber septum and the slurry is stirredat gentle reflux for 6 hours. After reaction, the fibrils are collectedand dried as in Preparation A.

Preparation H Preparation of Fibril-OH (Oxygenated derivative containingonly OH functionalization)

One half g of lithiated fibrils prepared in Preparation F aretransferred with dry, deoxygenated heptane in an argon-atmosphere glovebag to a 50 cc single neck flask fitted with a stopcock and magneticstirring bar. The flask is removed from the glove bag and stirred on amagnetic stirrer. The stopcock is then opened to the air and the slurrystirred for 24 hours. At the end of that time, the fibrils are separatedby filtration and washed with aqueous MeOH, and dried at 50° C. at 5"vacuum. The concentration of OH groups is determined by reaction with astandardized solution of acetic anhydride in dioxane (0.252M) at 80° C.to convert the OH groups to acetate esters, in so doing, releasing 1equivalent of acetic acid/mole of anhydride reacted. The total acidcontent, free acetic acid and unreacted acetic anhydride, is determinedby titration with 0.100N NaOH to an endpoint at pH 7.5.

Preparation I Preparation of Fibril-NH₂

One gram of thallated fibrils is prepared as in Preparation G. Thefibrils are slurried in dioxane and 0.5 g triphenyl phosphine dissolvedin dioxane is added. The slurry is stirred at 50° C. for severalminutes, followed by addition at 50° C. of gaseous ammonia for 30 min.The fibrils are then separated by filtration, washed in dioxane, then DIwater and dried at 80° C. at 5" vacuum. The amine concentration isdetermined by reaction with excess acetic anhydride and back-titrationof free acetic acid and unreacted anhydride with 0.100N NaOH.

4. DERIVATIZED POLYNUCLEAR AROMATIC, POLYHETERONUCLEAR AROMATIC ANDPLANAR MACROCYCLIC COMPOUNDS

The graphitic surfaces of fibrils allow for physical adsorption ofaromatic compounds. The attraction is through van der Waals forces.These forces are considerable between multi-ring heteronuclear aromaticcompounds and the basal plane carbons of graphitic surfaces. Desorptionmay occur under conditions where competitive surface adsorption ispossible or where the adsorbate has high solubility.

Preparation J Adsorption of Porphyrins and Phthalocyanines onto Fibrils

The preferred compounds for physical adsorption on fibrils arederivatized porphyrins or phthalocyanines which are known to adsorbstrongly on graphite or carbon blacks. Several compounds are available,e.g., a tetracarboxylic acid porphyrin, cobalt (II) phthalocyanine ordilithium phthalocyanine. The latter two can be derivatized to acarboxylic acid form.

The loading capacity of the porphyrin or phthalocyanines can bedetermined by decoloration of solutions when they are addedincrementally. The deep colors of the solutions (deep pink for thetetracarboxylic acid porphyrin in MeOH, dark blue-green for the Co(II)or the dilithium phthalocyanine in acetone or pyridine) are dischargedas the molecules are removed by adsorption onto the black surface of thefibrils.

Loading capacities were estimated by this method and the footprints ofthe derivatives were calculated from their approximate measurements(˜140 sq. Angstroms). For an average surface area for fibrils of 250 m²/g, maximum loading will be ˜0.3 mmol/g.

The tetracarboxylic acid porphyrin was analyzed by titration. Theintegrity of the adsorption was tested by color release in aqueoussystems at ambient and elevated temperatures.

The fibril slurries were initially mixed (Waring blender) and stirredduring loading. Some of the slurries were ultra-sounded after color wasno longer discharged, but with no effect.

After loading, Runs 169-11, -12, -14 and -19-1 (see Table II) werewashed in the same solvent to remove occluded pigment. All gave acontinuous faint tint in the wash effluent, so it was difficult todetermine the saturation point precisely. Runs 168-18 and -19-2 used thecalculated amounts of pigment for loading and were washed only verylightly after loading.

The tetracarboxylic acid porphyrin (from acetone) and the Cophthalocyanine (from pyridine) were loaded onto fibrils for furthercharacterization (Runs 169-18 and -19-2, respectively).

Analysis of Tetracarboxylic Acid Porphyrin

Addition of excess base (pH 11-12) caused an immediate pink colorationin the titrating slurry. While this did not interfere with thetitration, it showed that at high pH, porphyrin desorbed. The carboxylicacid concentration was determined by back titration of excess NaOH usingPh 7.5 as end-point. The titration gave a loading of 1.10 meq/g of acid,equivalent to 0.275 meq/g porphyrin.

Analysis of Cobalt or Dilithium Phthalocyanine

The concentrations of these adsorbates were estimated from decolorationexperiments only. The point where the blue-green tint did not fade after30 min was taken as the saturation-point.

A number of substituted polynuclear aromatic or polyheteronucleararomatic compounds were adsorbed on fibril surfaces. For adhesion, thenumber of aromatic rings should be greater than two per rings/pendantfunctional group. Thus, substituted anthracenes, phenanthrenes, etc.,containing three fused rings, or polyfuntional derivatives containingfour or more fused rings can be used in place of the porphyrin orphthalocayanine derivatives. Likewise, substituted aromatic heterocyclessuch as the quinolines, or multiply substituted heteroaromaticscontaining four or more rings can be used.

Table II summarizes the results of the loading experiments for the threeporphyrin/phthalocyanine derivatives.

                                      TABLE II    __________________________________________________________________________    Summary of Adsorption Runs                 Wgt.       Loading meq/g    EX.       RUN #            Adsorbate                 Fib, g Solv.                            g/g Form                                    Titration    __________________________________________________________________________    10A       169-11            TCAPorph                 19.6                     mg Acet                            0.18 g/g                                Acid                                    na    10B       169-12            TCAPorph                 33.3                     mg H.sub.2 O                            0.11                                Na Salt                                    na    10C       169-14            DiLiPhth                 119.0                     mg Acet                            0.170                                Li  na    10D       16g-19-1            CoPhth                 250.0                     mg Pyr 0.187                                Co  0.335 (cal)    10E       169-18            TCAPorph                 1.00                     g  Acet                            0.205                                Acid                                    1.10 (T)    10F       169-19-2            CoPhth                 1.40                     g  Pyr 0.172                                Co  0.303 (cal)    __________________________________________________________________________     TCAPorph = Tetracarboxylic Acid Porphyrin (cal) = calculated     DiLiPhth = Dilithium Phthalocyanine (T) = Titration     CoPhth = Cobalt (II) Phthalocyanine

5. CHLORATE OR NITRIC ACID OXIDATION

Literature on the oxidation of graphite by strong oxidants such aspotassium chlorate in conc. sulfuric acid or nitric acid, includes R. N.Smith, Quarterly Review 13, 287 (1959); M. J. D. Low, Chem. Rev. 60, 267(1960)). Generally, edge carbons (including defect sites) are attackedto give mixtures of carboxylic acids, phenols and other oxygenatedgroups. The mechanism is complex involving radical reactions.

Preparation K Preparation of Carboxylic Acid-Functionalized FibrilsUsing Chlorate.

The sample of CC fibrils was slurried in conc. H₂ SO₄ by mixing with aspatula and then transferred to a reactor flask fitted with gasinlet/outlets and an overhead stirrer. With stirring and under a slowflow of argon, the charge of NaClO₃ was added in portions at RT over theduration of the run. Chlorine vapors were generated during the entirecourse of the run and were swept out of the reactor into a aqueous NaOHtrap. At the end of the run, the fibril slurry was poured over crackedice and vacuum filtered. The filter cake was then transferred to aSoxhlet thimble and washed in a Soxhlet extractor with DI water,exchanging fresh water every several hours. Washing was continued untila sample of fibrils, when added to fresh DI water, did not change the pHof the water. The fibrils were then separated by filtration and dried at100° C. at 5" vacuum overnight.

The carboxylic acid content was determined by reacting a sample withexcess 0.100N NaOH and back-titrating with 0.100^(n) HCl to an endpointat pH 7.5. The results are listed in Table III.

                                      TABLE III    __________________________________________________________________________    Summary of Direct Oxidation Runs           Components, g                        Time     Rec Acid,    Ex.       RUN #           Fibrils               NaClO.sub.3 cc                    H.sub.2 SO.sub.4                        hours                            Wash Ph                                 Wgt meq/g    __________________________________________________________________________    11A       168-30           10.0               8.68 450 24  5.7  10.0                                     0.78    11B       168-36           12.0               13.9 600 24  5.9  13.7                                     0.75    __________________________________________________________________________

Preparation L Preparation of Carboxylic Acid-Functionalized FibrilsUsing Nitric Acid.

A weighed sample of fibrils was slurried with nitric acid of theappropriate strength in a bound bottom multi-neck indented reactor flaskequipped with an overhead stirrer and a water condenser. With constantstirring, the temperature was adjusted and the reaction carried out forthe specified time. Brown fumes were liberated shortly after thetemperature exceeded 70° C., regardless of acid strength. After thereaction, the slurry was poured onto cracked ice and diluted with DIwater. The slurry was filtered and excess acid removed by washing in aSoxhlet extractor, replacing the reservoir with fresh DI water everyseveral hours, until a slurried sample gave no change in Ph from DIwater. The fibrils were dried at 100° C. at 5" vacuum overnight. Aweighed portion of fibrils was reacted with standard 0.100N NaOH and thecarboxylic acid content determined by back-titration with 0.100N HCl.Surface oxygen content was determined by XPS. Dispersibility in waterwas tested at 0.1 wt % by mixing in a Waring Blender at high for 2 min.Results are summarized in Table IV.

                                      TABLE IV    __________________________________________________________________________    Summary of Direct Oxidation Runs    COMPONENTS    Gms.   cc   Acid                    Temp.   Wgt.                               COOH                                   ESCA, at % Disp    Ex.       Fibrils           Acid Conc.                    °C.                        Time                            Loss                               meq/g                                   C  O  H2O    __________________________________________________________________________    12A        1 (BN)           300     70%                    RT  24                          hr                            0  <0.1                                   98 2  P    12B        1 (BN)           300  15  rflx                        48  <5%                               <0.1                                   not analyzed                                         P    12C       20 (BN)           1.0              1 70  rflx                        7   25%                               0.8 not analyzed                                         G    12D       48 (BN)           1.0              1 70  rflx                        7   20%                               0.9 not analyzed                                         G    __________________________________________________________________________     P = Poor;     G = Good

6. SECONDARY DERIVATIVES OF FUNCTIONALIZED FIBRILS

Carboxylic Acid-functionalized Fibrils

The number of secondary derivatives which can be prepared from justcarboxylic acid is essentially limitless. Alcohols or amines are easilylinked to acid to give stable esters or amides. If the alcohol or amineis part of a di- or poly-functional molecule, then linkage through theO- or NH- leaves the other functionalities as pendant groups. Typicalexamples of secondary reagents are:

    ______________________________________                  PENDANT    GENERAL FORMULA                  GROUP     EXAMPLES    ______________________________________    HO--R, R = alkyl, aralkyl,                  R--       Methanol, phenol, tri-    aryl, fluoroethanol,    fluorocarbon, OH-terminated    polymer, SiR'.sub.3     Polyester, silanols    H.sub.2 N--R R = same as                  R--       Amines, anilines, fluorinated    above                   amines, silylamines, amine                            terminated polyamides    Cl--SiR.sub.3 SiR.sub.3 --                            Chlorosilanes    HO--R--OH, R = alkyl,                  HO--      Ethyleneglycol, PEG, Penta-    aralkyl, CH.sub.2 O--   erythritol, bis-Phenol A    H.sub.2 N--R--NH.sub.2, R = alkyl,                  H.sub.2 N--                            Ethylenediamine, polyethyl-    aralkyl                 eneamines    X--R--Y, R = alkyl, etc;                  Y--       Polyamine amides,    X = OH or NH.sub.2 ; Y = SH,                            Mercaptoethanol    CN, C═O, CHO, alkene,    alkyne, aromatic,    heterocycles    ______________________________________

The reactions can be carried out using any of the methods developed foresterifying or aminating carboxylic acids with alcohols or amines. Ofthese, the methods of H. A. Staab, Angew. Chem. Internat. Edit., (1),351 (1962) using N,N'-carbonyl diimidazole (CDI) as the acylating agentfor esters or amides, and of G. W. Anderson, et al., J. Amer. Chem. Soc.86, 1839 (1964), using N-Hydroxysuccinimide (NHS) to activate carboxylicacids for amidation were used.

Preparation M Preparation of Secondary Derivatives of FunctionalizedFibrils

N, N'-Carbonyl Diimidazole

Clean, dry, aprotic solvents (e.g., toluene or dioxane) are required forthis procedure. Stoichiometric amounts of reagents are sufficient. Foresters, the carboxylic acid compound is reacted in an inert atmosphere(argon) in toluene with a stoichiometric amount of CDI dissolved intoluene at R.T. for 2 hours. During this time, CO₂ is evolved. After twohours, the alcohol is added along with catalytic amounts of Na ethoxideand the reaction continued at 80° C. for 4 hr. For normal alcohols, theyields are quantitative. The reactions are:

1. R--COOH+Im--CO--Im→R--CO--Im+HIm+CO₂, Im=Imidazolide, HIm=Imidazole##STR19##

Amidation of amines occurs uncatalyzed at RT. The first step in theprocedure is the same. After evolution of CO₂, a stoichiometric amountof amine is added at RT and reacted for 1-2 hours. The reaction isquantitative. The reaction is:

3. R--CO--Im+R'NH₂ →R--CO--NHR+HIm

N-Hydroxysuccinimide

Activation of carboxylic acids for amination with primary amines occursthrough the N-hydroxysuccinamyl ester; carbodiimide is used to tie upthe water released as a substituted urea. The NHS ester is thenconverted at RT to the amide by reaction with primary amine. Thereactions are:

1. R--COOH+NHS+CDI→R--CONHS+Subst. Urea

2. R--CONHS+R'NH₂ →R--CO--NHR'

Silylation

Trialkylsilylchlorides or trialkylsilanols react immediately with acidicH according to:

R--COOH+Cl--SiR'₃ →R--CO--SiR'₃ +HCl

Small amounts of Diaza-1,1,1-bicyclooctane (DABCO) are used ascatalysts. Suitable solvents are dioxane and toluene.

Preparation N Preparation of Ester/Alcohol Derivatives from CarboxylicAcid-Functionalized Fibrils

The carboxylic acid functionalized fibrils were prepared as inPreparation K. The carboxylic acid content was 0.75 meq/g. Fibrils werereacted with a stoichiometric amount of CDI in an inert atmosphere withtoluene as solvent at R.T. until CO₂ evolution ceased. Thereafter, theslurry was reacted at 80° C. with a 10-fold molar excess ofpolyethyleneglycol (MW 600) and a small amount of NaOEt as catalyst.After two hours reaction, the fibrils were separated by filtration,washed with toluene and dried at 100° C.

Preparation O Preparation of Amide/Amine Derivatives from CarboxylicAcid-Functionalized Fibrils (177-041-1)

0.242 g of chlorate-oxidized fibrils (0.62 meq/g) was suspended in 20 mlanhydrous dioxane with stirring in a 100 ml RB flask fitted with a serumstopper. A 20-fold molar excess of N-Hydroxysuccinimide (0.299 g) wasadded and allowed to dissolve. This was followed by addition of 20-foldmolar excess of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC)(0.510 g), and stirring was continued for 2 hr at RT. At the end of thisperiod stirring was stopped, and the supernatant aspirated and thesolids were washed with anhydrous dioxane and MeOH and filtered on a0.45 micron polysulfone membrane. The solids were washed with additionalMeOH on the filter membrane and vacuum-dried until no further weightreduction was observed. Yield of NHS-activated oxidized fibrils was 100%based on the 6% weight gain observed.

100 μl ethylenediamine (en) was added to 10 ml 0.2M NaHCO₃ buffer. Anequivalent volume of acetic acid (HOAC) was added to maintain the pHnear 8. NHS-activated oxidized fibrils (0.310 g) was added with vigorousstirring and reacted for 1 hr. An additional 300 μl of en and 300 μlHOAc was added for an additional 10 min. The solution was filtered on0.45 micron polysulfone membrane and washed successively with NaHCO₃buffer, 1% HCl, DI water and EtOH. The solids were dried under vacuoovernight. The HCl salt was converted back to the free amine by reactionwith NaOH (177-046-1) for further analysis and reactions.

ESCA was carried out to quantify the amount of N present on the aminatedfibrils (GF/NH₂). ESCA analysis of 177-046-1 showed 0.90 at % N(177-059). To further assess how much of this N is present as bothaccessible and reactive amine groups, a derivative was made by the gasphase reaction with pentafluorobenzaldehyde to produce the correspondingSchiff Base linkages with available primary amine groups. ESCA analysisstill showed the 0.91 at % N, as expected, and 1.68 at % F. Thistranslates into a 0.34 at % of N present as reactive primary amine onthe aminated fibrils (5 F per pentafluorobenzaldehyde molecule). A levelof 0.45 at % N would be expected assuming complete reaction with thefree ends of each N. The observed level indicates a very high yield fromthe reaction of N with NHS-activated fibril and confirms the reactivityof the available free amine groups.

At the level of 0.34 at % N present as free amine calculated from theESCA data, there would be almost complete coverage of the fibrils by thefree amine groups allowing coupling of other materials.

Preparation P Preparation of Silyl Derivative from CarboxylicAcid-Functionalized Fibrils

Acid functionalized fibrils prepared as in Preparation K were slurriedin dioxane in an inert atmosphere. With stirring, a stoichiometricamount of chlorotriethyl silane was added and reacted for 0.5 hr, afterwhich several drops of a 5% solution of DABCO in dioxane was added. Thesystem was reacted for an additional hour, after which the fibrils werecollected by filtration and washed in dioxane. The fibrils were dried at100° C. in V" vacuum overnight.

Table V summarizes the secondary derivative preparations. The productswere analyzed by ESCA for C, O, N, Si and F surface contents.

                                      TABLE V    __________________________________________________________________________    Summary of Secondary Derivative Preparations                         ESCA ANALYSIS, ATOM %    REACTANT PENDANT GROUP                         S  C  N  O Si                                      F    __________________________________________________________________________    As Grown --          -- 98.5                               -- 1.5                                    --                                      --    Chlorate Oxidized             --COOH, C═O, C--OH                         -- 92.4                               -- 7.6                                    --                                      --    H.sub.2 N--C.sub.2 H.sub.4 --NH.sub.2             --CONHC.sub.2 H.sub.4 NH.sub.2                         -- 99.10                               0.90                                  --                                    --                                      --             --CONHC.sub.2 H.sub.4 N = OC.sub.6 F.sub.5                         -- 97.41                               0.91                                  --                                    --                                      1.68    __________________________________________________________________________

Preparation Q Preparation of silyl Derivative from CarboxylicAcid-Functionalized Fibrils

Acid functionalized fibrils prepared as in Preparation K are slurried indioxane in an inert atmosphere. With stirring, a stoichiometric amountof chlorotriethyl silane is added and reacted for 0.5 hr, after whichseveral drops of a 5% solution of DABCO in dioxane is added. The systemis reacted for an additional hour, after which the fibrils are collectedby filtration and washed in dioxane. The fibrils are dried at 100° C. in5" vacuum overnight.

Table VI summarizes the secondary derivative preparations. Products areanalyzed by ESCA. The analysis confirms the incorporation of the desiredpendant groups. The products are analyzed by ESCA for C, O, N, Si and Fsurface contents.

                  TABLE VI    ______________________________________    Summary of Secondary Derivative Preparations                      ESCA ANALYSIS, ATOM %    REACTANT  PENDANT GROUP S     C   N   O   Si  F    ______________________________________    CF.sub.3 CH.sub.2 OH              --COOCH.sub.2 CF3                            NOT ANALYZED    PolyEG-600              --CO--(OC.sub.2 H.sub.4 O--)H                            NOT ANALYZED    HO--C.sub.2 H.sub.4 --SH              --COOC.sub.2 H4SH    Cl--SiEt.sub.3              --COSiEt.sub.3    ______________________________________

Sulfonic Acid-Functionalized Fibrils

Aryl sulfonic acids, as prepared in Preparation A can be further reactedto yield secondary derivatives. Sulfonic acids can be reduced tomercaptans by LiAlH₄ or the combination of triphenyl phosphine andiodine (March, J.P., p. 1107). They can also be converted to sulfonateesters by reaction with dialkyl ethers, i.e.,

Fibril-SO₃ H+R--O--R→Fibril-SO₂ OR+ROH

Fibrils Functionalized by Electrophilic Addition to Oxygen-Free FibrilSurfaces or by Metallization

The primary products obtainable by addition of activated electrophilesto oxygen-free fibril surfaces have pendant --COOH, --COCl, --CN, --CH₂NH₂, --CH₂ OH, --CH₂ -- Halogen, or HC═O. These can be converted tosecondary derivatives by the following:

Fibril-COOH→see above. Fibril-COCl (acid chloride)+HO--R--Y→F--COO--R--Y(Sec. 4/5)

Fibril-COCl+NH₂ --R--Y→F--CONH--R--Y

Fibril-CN+H₂ →F--CH₂ --NH₂

Fibril-CH₂ NH₂ +HOOC--R--Y→F--CH₂ NHCO--R--Y

Fibril-CH₂ NH₂ +O═CR--R'Y→F--CH₂ N═CR--R'--Y

Fibril-CH₂ OH+O(COR--Y)₂ →F--CH₂ OCOR--Y

Fibril-CH₂ OH+HOOC--R--Y→F--CH₂ OCOR--Y

Fibril-CH₂ -Halogen+Y⁻ →F--CH₂ --Y+X⁻ Y⁻ =NCO³¹ , --OR ⁻ Fibril-C═O+H₂N--R--Y→F--C═N--R--Y

Fibrils Functionalized by Adsorption of Polynuclear or PolyheteronuclearAromatic or Planar Macrocyclic Compounds

Dilithium phthalocyanine: In general, the two Li⁺ ions are displacedfrom the phthalocyanine (Pc) group by most metal (particularlymulti-valent) complexes. Therefore, displacement of the Li⁺ ions with ametal ion bonded with non-labile ligands is a method of putting stablefunctional groups onto fibril surfaces. Nearly all transition metalcomplexes will displace Li⁺ from Pc to form a stable, non-labilechelate. The point is then to couple this metal with a suitable ligand.

Cobalt (II) Phthalocyanine

Cobalt (II) complexes are particularly suited for this. Co⁺⁺ ion can besubstituted for the two Li⁺ ions to form a very stable chelate. The Co⁺⁺ion can then be coordinated to a ligand such as nicotinic acid, whichcontains a pyridine ring with a pendant carboxylic acid group and whichis known to bond preferentially to the pyridine group. In the presenceof excess nicotinic acid, Co(II)Pc can be electrochemically oxidized toCo(III)Pc, forming a non-labile complex with the pyridine moiety ofnicotinic acid. Thus, the free carboxylic acid group of the nicotinicacid ligand is firmly attached to the fibril surface.

Other suitable ligands are the aminopyridines or ethylenediamine(pendant NH₂), mercaptopyridine (SH), or other polyfunctional ligandscontaining either an amino- or pyridyl- moiety on one end, and anydesirable function on the other.

7. 3-DIMENSIONAL STRUCTURES

The oxidized fibrils are more easily dispersed in. aqueous media thanunoxidized fibrils. Stable, porous 3-dimensional structures with meso-and macropores (pores >2 nm) are very useful as catalysts orchromatography supports. Since fibrils can be dispersed on anindividualized basis, a well-dispersed sample which is stabilized bycross-links allows one to construct such a support. Functionalizedfibrils are ideal for this application since they are easily dispersedin aqueous or polar media and the functionality provides cross-linkpoints. Additionally, the functionality provides points to support thecatalytic or chromatographic sites. The end result is a rigid,3-dimensional structure with its total surface area accessible withfunctional sites on which to support the active agent.

Typical applications for these supports in catalysis include their useas a highly porous support for metal catalysts laid down byimpregnation, e.g., precious metal hydrogenation catalysts. Moreover,the ability to anchor molecular catalysts by tether to the support viathe functionality combined with the very high porosity of the structureallows one to carry out homogeneous reactions in a heterogeneous manner.The tethered molecular catalyst is essentially dangling in a continuousliquid phase, similar to a homogeneous reactor, in which it can make useof the advantages in selectivities and rates that go along withhomogeneous reactions. However, being tethered to the solid supportallows easy separation and recovery of the active, and in many cases,very expensive catalyst.

These stable, rigid structures also permits carrying out heretofore verydifficult reactions, such as asymmetric syntheses or affinitychromatography by attaching a suitable enantiomeric catalyst orselective substrate to the support. Derivatization through Metallo-Pc orMetallo-porphyrin complexes also allows for retrieval of the ligandbonded to the metal ion, and furthermore, any molecule which is bondedto the ligand through the secondary derivatives. For example, in thecase where the 3-dimensional structure of functionalized fibrils is anelectrode, or part of an electrode, and the functionalization hasresulted from adsorption of Co(II)Pc, electrochemical oxidation ofCo(II) to Co(III) in the presence of nicotinic acid will produce anon-labile Co(III)-pyridyl complex with a carboxylic acid as the pendentgroup. Attaching a suitable antigen, antibody, catalytic antibody, orother site-specific trapping agent will permit selective separations ofmolecules (affinity chromatography) which are otherwise very difficultto achieve. After washing the electrode to remove occluded material, theCo(III) complex containing the target molecule can be electrochemicallyreduced to recover the labile Co(II) complex. The ligand on Co(II)containing the target molecule can then be recovered by mass actionsubstitution of the labile CO(II) ligand, thereby effecting a separationand recovery of molecules which are otherwise very difficult orexpensive to perform (e.g., chiral drugs).

Another example of 3-dimensional structures are fibril-ceramiccomposites.

Preparation R Preparation of Alumina-Fibril Composites (185-02-01)

One g of nitric acid oxidized fibrils (185-01-02) was highly dispersedin 100 cc DI water using and U/S disintegrator. The fibril slurry washeated to 90° C. and a solution of 0.04 mol aluminum tributoxidedissolved in 20 cc propanol was slowly added. Reflux was continued for 4hr, after which the condenser was removed to drive out the alcohol.After 30 min the condenser was put back and the slurry refluxed at 100°C. overnight. A black sol with uniform appearance was obtained. The solwas cooled to RT and after one week, a black gel with a smooth surfacewas formed. The gel was heated at 300° C. in air for 12 hr.

The alumina-fibril composites were examined by SEM. Micrographs ofcracked surfaces showed a homogeneous dispersion of fibrils in the gel.

Preparation S Preparation of Silica-Fibril Composites (173-85-03)

Two g of nitric acid oxidized fibrils (173-83-03) were highly dispersedon 200 cc ethanol using ultrasonification. A solution of 0.1 moltetraethoxysilane dissolved in 50 cc ethanol was slowly added to theslurry at RT, followed by 3 cc conc. HCL. The mixture was heated to 85°C. and maintained at that temperature until the volume was reduced to100 cc. The mixture was cooled and set aside until it formed a blacksolid gel. The gel was heated at 300° C. in air.

The silica-fibril composites were examined by SEM. Micrographs ofcracked surfaces showed a homogeneous dispersion of fibrils in the gel.

Similar preparations with other ceramics, such as zirconia, titania,rare earth oxides as well as ternary oxides can be prepared.

As illustrated by the foregoing description and examples, the inventionhas application in the formulation of a wide variety of functionalizednanotubes.

The terms and expressions which have been employed are used as terms ofdescription and not of limitations, and there is no intention in the useof such terms or expressions of excluding any equivalents of thefeatures shown and described as portions thereof, its being recognizedthat various modifications are possible within the scope of theinvention

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

While a wide range of nanotubes can be employed in the assays of theinvention, generally the nanotubes have a density of from 1.0 to 5.0g/mL and preferably have a density of from 1.1 to 2 g/mL. Choice of theoptimum density is within the skill of the art, the rate of settling ingravity-driven assays being a trade-off between the speed of the assayand the desire to create a uniform layer of complex on the electrodesurface.

Nanotubes having a wide range of mean diameters can also be employed.Particles having a mean diameter of from 0.001 to 100 μm can be used andpreferably the particles have a mean diameter of from 0.01 to 10 μm.Lengths of the nanotubes are at least five times the diameter.

Wide ranges of concentration of particles in the assay composition canalso be employed. For example, the concentration can range from 1×10⁻⁹to 1×10⁻² g/mL to preferably from 1×10⁻⁸ to 1×10⁻³ g/mL. Desirably, thedensity of the particles, their size and their concentration is selectedsuch that the particles settle at a rate of at least 0.5 mm/min andpreferably at a faster rate.

In the filtration mode of performing the invention, the filtration meansdesirably has a pore size, measured as mean diameter, from broadly 0.01to 90% of the mean diameter of the particles and preferably from 10% to90% of that diameter.

The nanotubes may be paramagnetic or ferromagnetic and may be coatedwith various materials to which binding compounds are coupled so thatthe magnetic particle can be used in assays. Desirably the magneticnanotubes used in the invention have a susceptibility of at least 0.001cgs units and desirably the susceptibility is at least 0.01 cgs units.The magnetic nanotubes may have a broad range of densities, i.e. fromsubstantially less than that of water, 0.01, to 5 g/mL and preferablyfrom 0.5 to 2 g/mL. The particle sizes can range from 0.001 to 100 μmand preferably from 0.01 to 10 μm. The concentration of the particlesmay range broadly from 1×10⁻⁹ to 1×10⁻² g per mL and preferably is from1×10⁻⁸ to 1×10⁻³ g per mL.

Desirably the magnetic nanotubes which are used have a low magneticremanence, as described for example EP 0,180,384, so that after themagnetic field is removed from the electrode surface, the nanotubesdemagnetize and can be swept out of the assay cell. Desirably thedensity, concentration and size of the magnetic nanotubes is chosen suchthat the settling time is at least 0.5 mm/min and desirably it is abovethat rate. In operation of the magnetic cell it is often desirable toremove the magnet means from the electrode surface prior to inducingelectrochemiluminescence in order not to interfere with the operation ofthe photomultiplier tube.

Assays

A variety of assays can be performed using the methods of the invention.An assay was conducted using functionalized carbon nanotubes. The assayinvolved an ECL detection of hydrolytic enzymes using carbon nanotubes(fibrils). Carbon nanotubes (fibrils) were chemically modified withsubstrates of hydrolytic enzymes. On the end of the substrate farthestfrom the fibril was attached a derivative of Ru(bpy)₃ ²⁺. The generalstructure of the solid phase was as follows: fibril-substrate (scissilebond)-Ru(bpy)₃ ²⁺. If an enzyme is present that cleaves the scissilebond, the Ru(bpy)₃ ²⁺ end of the substrate is released into solution bythe action of the enzyme. Following mixture and incubation of thefibrils with the enzyme, the fibrils are removed from the solution (byfiltration or centrifugation). The ECL of the remaining solution ismeasured. If the enzyme was present, the Ru(bpy)₃ ²⁺ end of thesubstrate will be present in the solution and will emit light. Thus,presence of the specific enzyme results in light emission from thesolution phase of the mixture. The assay is a novel ECL assay forproteases because it does not involve antibodies (it is not animmunoassay). Thus, it has the advantage of being an assay for enzymeactivity rather than an assay for the enzyme's presence (the enzyme maybe inactive). Moreover, the assay uses fibrils as a solid support.Fibrils are attractive because of their high surface area andamenability in attaching biomolecules such as enzyme substrates. Inaddition, the functionalized fibrils may be formed into a flow throughmembrane (mat). The enzyme mixture could rapidly flow through to releaseRu(bpy)₃ ²⁺ and make the assay rapid and convenient.

A DNA probe assay using fibrils and ECL was conducted. Carbon nanotubes(fibrils) were used as a solid support in DNA probe assays as aseparation media to separate analyte from complex mixtures which mayinclude biological fluids and added reagents. Fibrils were modified withavidin (or streptavidin), either by covalent attachment (via NHS ester)or by adsorption to alkyl fibrils. Biotinylated ssDNA (the "analyte")bound to the avidin fibrils and was detected by the ECL of acomplementary single stranded oligonucleotide which had been labeledwith Ru(bpy)₃ ²⁺.

One format for detecting a natural (non-biotinylated) DNA fragment is acompetitive format where the Ru(bpy)₃ ²⁺ -labeled oligo can bind toeither the natural DNA or to an introduced biotinylated DNA. Thus, themore analyte that is present (non-biotinylated DNA), the less labeledoligo remains to bind to the biotinylated DNA, which when captured onavidin fibrils gives an ECL signal.

This represents the first time carbon nanotubes have been used in DNAanalysis. The advantages are: (1) carbon nanotubes have a very highsurface area which means that less solid support is necessary than withother solid supports. (2) Fibrils are electrically conductive, which isan attractive property for a solid support in ECL applications, in whichthe solid support rests against an electrode. Being electricallyconductive, more of the surface area is in electrochemical contact thanwith other supports which are not conductive.

An ECL-based immunoassay using immobilized antibodies on carbonnanotubes (fibrils) would be advantageous. Antibody fibrils could beused in ECL applications in a competitive immunoassay format (wherethere is a competition between the analyte and Ru(bpy)₃ ²⁺ -labeledanalyte for binding to the antibody fibrils) or in a sandwich format(where a Ru(bpy)₃ ²⁺ -labeled secondary antibody binds to the antibodyfibril-analyte complex). In both cases, ECL light emission signals thepresence or absence of the analyte of interest. Antibody can beimmobilized on fibrils by several different methods, by covalent ornon-covalent means. For non-covalent immobilization, antibody isadsorbed onto unmodified fibrils or onto fibrils modified to enhanceadsorption characteristics. Such modified fibrils have hydrophobicappendages (alkyl chains or phenyl-alkyl chains). Covalentimmobilization of antibodies onto fibrils can be achieved by threedifferent methods: by NHS ester activation of carboxylated fibrils, byreductive amination of antibody carbohydrate groups, and bysulfhydryl/maleimido fibrils reacting with reduced or maleimido-modifiedantibodies. The immobilization of antibodies on carbon fibrils isadvantageous. Antibody fibrils have a number of unique propertiescompared with other solid supports for antibodies. These advantagesinclude high surface area per weight, electrical conductivity(especially in ECL applications), and chemical and physical stability.

Carbon nanotubes were used as ECL-based biosensors. Bifunctional fibrilswere prepared wherein a derivative of the enzyme cofactor NAD+ wasattached to one functional group and a derivative of Ru(bpy)₃ ²⁺ wasattached to the other functional group (COOH). The biosensor fibrilswere mixed with a solution that contained the analyte (a dehydrogenase,in this case G6PDH) and the substrate of the dehydrogenase was added (inthis case, glucose-6-phosphate). Following a time suitable for theenzyme to react with its substrate and convert the NAD+ groups on thefibrils to NADH, the ECL of the fibrils were measured in an ECLinstrument. A change in the ECL properties of the biosensor fibrilsindicated the presence of the enzyme, G6PDH. The following advantageswere found: (1) The close proximity of the NAD+ and Ru(bpy)₃ ²⁺coreactants results in an intramolecular ECL reaction which is moreefficient than intermolecular ECL reactions. (2) The ECL active reagents(the NAD+ and Ru(bpy)₃ ²⁺ coreactants) are immobilized on fibrils, whichallows them to settle or be magnetically drawn to the ECL electrode,resulting in enhanced light emission. (3) Fibrils have an extremely highsurface area, such that large amounts of the coreactants (NAD+ andRu(bpy)₃ ²⁺ can be immobilized, theoretically enhancing light outputduring detection of dehydrogenases. (4) Fibrils are electricallyconductive - when the biosensors reach the electrode, much of theirsurface area which is not in actual contact with the electrode canreceive voltage and participate in ECL reactions. (5) The biosensor isdesigned to detect dehydrogenases that use either NAD+ or NADH as acofactor. Many of these enzymes exist and could be detected using thisinvention.

Carbon fibrils were used as a solid support for an enzyme biosensor.Because the enzyme biosensor is water soluble, it can only be used once,unless it is immobilized (immobilization allows it to be recovered fromthe analyte solution). U.S. application Ser. No. 08/467,712 discussesand claims immobilization to a solid support and to a (solid) electrode.The present application demonstrates the use of carbon fibrils as thesolid support. The enzyme biosensor can be attached to fibrils byadsorption. The fibrils may either be unmodified fibrils or, preferably,chemically modified fibrils. Chemical modification of the fibrils ispreferably by alkylation. Adsorption of the biosensor is carried out byincubation with mixing of the enzyme biosensor and the alkylatedfibrils. Fibrils as a solid support are novel and attractive because:(1) they have a high surface area of immobilization resulting inpotentially high light emission, (2) proteins such as the biosensor canbe conveniently adsorbed without covalent chemistry, (3) fibrils areelectrically conductive which may enhance ECL when in contact with anelectrode, (4) fibrils themselves may be made into electrodes, in whichcase the electrode itself if a biosensor support.

Magnetically-susceptible particles are useful as devices forseparations. Separations of, for example, a specific analyte from acomplex mixture are useful in the diagnostics industry where an analytemay be present in a complex mixture such as blood. Because the complexmixture may interfere with the analysis of the analyte, it is desirableto bind the analyte to a solid surface so that the complete mixture canbe washed away from the analyte, thus allowing its determination. If thedesired analyte can specifically bind to a magnetically-susceptibleparticle, that particle can be held by a magnet, allowing washing of thecomplex mixture away from the analyte, which can then be detected.Because fibrils are particles that can specifically and efficiently bindbiomolecules, magnetically-susceptible fibrils advantageously are usedas solid phase separation devices, especially in ECL analyses. Fibrilswere made magnetically-susceptible by performing treatments, such aschemical reactions or changes of the solvent that they are suspended in(for example, from water to DMF). It is speculated that suchmanipulations cause changes in the physical aggregation of multiples offibrils. Because fibrils have some (approximately 0.5% by weight) ironin them, they have the ability, once aggregated, to be substantiallymagnetically susceptible. Several treatments were carried out whichincrease the magnetic susceptibility of fibrils. These treatments arepostulated to cause changes in the aggregation of fibrils, which inturn, cause them to be efficiently drawn to a magnet.Magnetically-susceptible fibrils have at least two unique advantages.First, they have a very high surface area per weight. Thus, less fibrilsneed to be used (as compared to for example Dynal beads) to achieve thesame result. Secondly, fibrils are electrically conductive. Thus, theyhave an advantage in applications such as ECL where the magneticallysusceptible particles are held to an electrode. Thus, being electricallyconductive, they actually become part of the electrode, enhancing thelight output efficiency.

EXAMPLES

(1) Instrumentation

A flow-through apparatus, employing three electrodes, as described inFIGS. 1 and 2, was used.

Working Electrode--Au disk, 3 mm diameter

Counter Electrode--Au disk, 3 mm diameter

Reference Electrode--Ag/AgCl

Teflon Gasket (0.15"thick)

Plexiglas Faceplate

Inlet Tubing=0.042" id polypropylene

Aspiration Rates:variable from 0.01 to 5 mL/min

Potentiostat: microprocessor controlled Luminometer using Hamamatsu R374PMT (low gain red sensitive tube); PMT Voltage variable 0-1400 V

(2) ECL Measurement Cycle (three electrode cell operation)

The ECL measurement cycle consists of three steps:

(1) preconditioning, (2) measuring, and (3) cleaning. Thepreconditioning step involves the application of a voltage triangle waveof 0.0 V to +2.2 V to -1.0 V to +0.6 V at 2.0 V/sec. The measurementstep involves the application of a triangle waveform from +0.6 to +2.8 Vto +2.0 V at 1.0 V/s. The cleaning step involves the application of avoltage square wave from +0.0 V to +3.0 V to -0.5 V to 0.0 V. Allvoltages are relative to the Ag/AgCl reference electrode.

EXAMPLE 1

Ruthenium Tag Peptide Fibril Synthesis

This example shows the synthesis of an enzyme detection reagent in ECLAnalyzer. Ruthenium peptide fibrils synthesis: Tetrapeptide ofFmocNH-Gly-Lys(N.sup.ε -CBZ)-Phe-Gly-COOH was synthesized byconventional solution phase methods and this peptide (71 mg, 0.093 mmol)was reacted with a primary amine derivatized version of Ru(bpy)₃ ²⁺(IGEN, Inc., Gaithersburg, Md.) (73 mg, 0.078 mmol), EDC (17.8 mg, 0.093mmol) was used as a activating reagent, and HOBT (12.58 mg, 0.093 mmol)as a catalyst. The product FmocNH-Gly-Lys(NE-CBZ)-Phe-Gly-CO-NH-Tag (170mg, 0.223 mmol) was deprotected with piperidine (96 ml) and methylenechloride (1.1 ml). The structure of the tetrapeptide-Ru(bpy)₃ ²⁺compound was confirmed by 1H-NMR. To the solution oftetrapeptide-Ru(bpy)₃ ²⁺ (5 mg, 0.003 mmol) in methylene chloride (2 ml)was added carboxyl fibrils (54 mg). Then EDC (5.8 mg, 0.03 mmol) andHOBT (4 mg, 0.03 mmol) were added. The reaction mixture was stirredovernight. The fibrils were extensively washed with water, methanol,acetonitrile and methylene chloride. The product fibrils were treatedwith trimethylsilyl iodide (TMSI, 1 ml) in acetonitrile (4 ml) for 3hours at 40° C. The final product fibrils were extensively washed withwater, methanol, acetonitrile, IGEN standard ECL assay buffer (IGEN,Inc., Gaithersburg, Md.) and methylene chloride.

EXAMPLE 2 ECL Assay of Trypsin and Chymotrypsin Activity Using Ru(bpy)₃²⁺ -Labeled Peptide Fibrils

A Ru(bpy)₃ ²⁺ -labeled tetrapeptide (NH₂ -Gly-Lys-Phe-Gly-Ru(bpy)₃ ²⁺)was conjugated to carboxylated fibrils as described in Example 1. TheRu(bpy)₃ ²⁺ -labeled peptide fibrils (RPF) were used to detect theactivity of the hydrolytic enzymes trypsin and chymotrypsin. In brief,the RPF were added to an aqueous solution containing either or both ofthe enzymes. Because the peptide was designed to contain specificcleavage sites for both trypsin and chymotrypsin (FIG. 3), and becausefibrils are a solid, the action of the enzymes would liberate eitherPhe-Gly-Ru(bpy)₃ ²⁺ (trypsin) or Gly-Ru(bpy)₃ ²⁺ (chymotrypsin) into thesolution. Following a suitable incubation time for the enzymes to cleavethe solid phase peptide, the aqueous solution was separated from thefibrils by standard means such as centrifugation, filtration, or removalof the fibrils with a magnet. The liberated Phe-Gly-Ru(bpy)₃ ²⁺ orGly-Ru(bpy)₃ ²⁺ in the aqueous solution is then detected by ECL.

Although assays of trypsin and chymotrypsin are shown here, otherhydrolytic enzymes could be detected using fibrils conjugated to theappropriate Ru(bpy)₃ ²⁺ -labeled enzyme substrate. Such enzymes (andmodified fibrils) include: nucleases (using fibrils conjugated toRu(bpy)₃ ²⁺ -labeled RNA, single stranded DNA, or double stranded DNA),glycosidases (using fibrils conjugated to Ru(bpy)₃ ²⁺ -labeled sugars,oligosaccharides, or polysaccharides), or lipases (using fibrilsconjugated to Ru(bpy)₃ ²⁺ -labeled lipids).

EXAMPLE 3 ECL Detection of Trypsin Using Fibrils

To 2.97 ml suspensions of RPF (Fibrils-Gly-LysPhe-Gly-Ru(bpy)₃ ²⁺) (2.2mg/mL) in standard ECL Assay Buffer (IGEN, Inc., Gaithersburg, Md.) wasadded either 30 μL of 58.9 μM trypsin (final concentration=0.59 μM) in 1mM HCl or 30 μl of 1 mM HCl. The two suspensions were then rotated atroom temperature. Periodically, the rotation was stopped, the suspensionwas quickly centrifuged, and the ECL of the aqueous supernatant wasmeasured. The ECL results after 30 minutes of incubation showed that theECL ratios of the samples (ECL with trypsin/ECL without trypsin) was1.29. After 44 hours, the ECL ratio was 2.05. These results demonstratedthat trypsin could be detected by its ability to hydrolytically liberatean electrochemiluminescent ruthenium label from fibrils.

EXAMPLE 4 ECL Detection of Chymotrypsin Using Fibrils

To 2.97 ml suspensions of RPF (Fibrils-Gly-LysPhe-Gly-Ru(bpy)₃ ²⁺) (0.15mg/mL) in standard ECL Assay Buffer (IGEN, Inc., Gaithersburg, Md.) wasadded either 30 μl of 34.2 μM chymotrypsin (final concentration=0.34 μM)in 1 mM HCl or 30 μl of 1 mM HCl. The suspensions were rotated at roomtemperature. Periodically, the rotation was stopped, the suspension wasquickly centrifuged, and the ECL of the aqueous supernatant wasmeasured. The ECL results showed that initially (time=0) the ECL ratioof the samples (ECL with chymotrypsin/ECL without chymotrypsin) was1.06. After 30 minutes of incubation, the ratio rose to 1.25, and after23 hours of incubation, the ratio rose to 1.85. These data show thatchymotrypsin activity could be detected by the ability of the enzyme toliberate an electrochemiluminescent label, Ru(bpy)₃ ²⁺, from a fibrilsolid support.

EXAMPLE 5 Covalent Attachment of Proteins to Fibrils Via NHS Ester

To demonstrate that protein can be covalently linked to fibrils via NHSester, streptavidin, avidin and trypsin were attached to fibrils asfollows.

0.5 mg of NHS-ester fibrils were washed with 5 mM sodium phosphatebuffer (pH=7.1) and the supernatant was discarded. 200 μl streptavidinsolution (1.5 mg in the same buffer) was added to the fibrils and themixture was rotated at room temperature for 5.5 hours. The fibrils werethen washed with 1 ml of the following buffers in sequence: 5 mM sodiumphosphate (pH=7.1), PBS (0.1M sodium phosphate, 0.15M NaCl, pH=7.4),ORIGEN assay buffer (IGEN, Inc., Gaithersburg, Md.) and PBS. Thestreptavidin fibrils were stored in PBS buffer for further use.

2.25 mg NHS-ester fibrils were sonicated in 500 μl of 5 mM sodiumphosphate buffer (pH=7.1) for 40 minutes and the supernatant wasdiscarded. The fibrils were suspended in 500 μl of 5 mM sodium phosphatebuffer (pH=7.1) and 300 μl of avidin solution made in the same buffercontaining 2 mg avidin (Sigma, A-9390) was added. The mixture wasrotated at room temperature for two hours, stored at 4° C. overnight androtated at room temperature for another hour. The fibrils were washedwith 1 ml of 5 mM sodium phosphate buffer (pH=7.1) four times and PBSbuffer twice. The avidin fibrils were suspended in 200 μl PBS buffer forstorage.

Trypsin fibrils were prepared by mixing 1.1 mg NHS-ester fibrils(treated as in avidin fibrils) and 200 μl of 1.06 mM trypsin solutionmade in 5 mM sodium phosphate buffer (pH=7.1) and rotating at roomtemperature for 6.5 hours. The trypsin fibrils were then washed by 1 mlof 5 mM sodium phosphate buffer (pH=7.1) three times and suspended in400 μl of the same buffer for storage.

EXAMPLE 6 DNA Probe Assay Using ECL Analyzer

To eliminate non-specific binding of analyte on streptavidin (or avidin)bound fibrils in ECL assay, these fibrils were blocked by 4 mg/ml BSA(bovine serum albumin) solution for 6 hours at room temperature andovernight at 4° C.

The DNA probe assay is depicted in FIG. 4. The experiment was startedwith washing 115 μg of BSA blocked avidin-fibrils and streptavidinfibrils (from Example 5) twice with 5 mM sodium phosphate buffer(pH=7.1). Each fibril was aliquoted into two tubes with ˜57 μg in 100 μlof the same buffer. One tube of (strept)avidin fibrils was mixed with 4μl of 4 nM biotinylated DNA (70 nucleotides) that was bound to aruthenium tag labeled oligomer. The other tube was added with 4 μl ofruthenium tag labeled oligomer (same concentration as in thebiotinylated DNA sample) only for a control assay. The reaction mixtureswere incubated at room temperature for 15 minutes and washed seven timeswith 300 μl of ORIGEN assay buffer (IGEN, Inc., Gaithersburg, Md.). Thefibrils were then suspended in 600 μl ORIGEN assay buffer and aliquotedto two tubes for duplicate ECL counting, using gravity capture of thefibrils. The result of the ECL assay were summarized as follows:

    ______________________________________    sample              ECL count    ______________________________________    avidin fibrils (control)                         161    avidin fibrils (DNA probe)                        2212    streptavidin fibrils (control)                         885    streptavidin fibrils (DNA probe)                        4248    ______________________________________

The same assay of avidin fibrils was also carried out using magneticcapture of the fibrils. The experimental procedure was the same as aboveexcept for no washing after DNA binding to fibrils. The sample waswashed in the ECL analyzer with programmed steps. The ECL count was 3835for avidin fibril DNA probe verses 205 for the control.

Avidin adsorbed on C8-fibrils were also examined by the DNA probe assayusing magnetic capture of the fibrils as described above. The ECL countwas 15,792 for avidin fibril DNA probe verses 205 for the control.

EXAMPLE 7 Covalent Immobilization of Antibodies on Carbon Nanotubes

The object of the example is to immobilize antibodies on the surfaces ofcarbon nanotubes. Such antibody-modified nanotubes can be used invarious applications including immunoassay detection of specificanalytes and biospecific affinity separations. Immunoassays could becarried out using antibody-modified nanotubes as a solid support. Use ofa solid support allows for washing steps if the solid support is eitherfixed, or otherwise separated from the solution phase (by for instance,filtration, centrifugation, or magnetism). Use of wash steps wouldpermit the use of antibody-modified nanotubes in many types ofelectrochemiluminescence-based immunoassays. The antibody-modifiednanotubes could be used as replacements of the conventionally-usedmagnetic beads, or as a suspendible support capable of capture byfiltration, or as a permanently-fixed support ion a disposablecartridge.

Fibril-Antibody coupling Between Primary Amine and Carboxyl Groups

Formation of NHS Ester Fibrils

A suspension of 148.4 mg of carbon fibrils (Hyperion CatalysisInternational, Cambridge, Mass.) in a CH₂ Cl₂ /dioxane mixture was mixedwith 239 mg of N-hydroxysuccinimide (NHS) and 399 mg of EDC and thereaction was allowed to proceed for 4 hours. The yield of the resultingNHS ester-modified fibrils (NHS-Fibrils) was 214.7 mg.

Formation of Antibody-Modified Fibrils

NHS ester fibrils (84.5 mg) were pre-treated with 1.0 mL coupling buffer(0.2M NaHCO₃, pH 8.1), then suspended in another 1.0 mL of couplingbuffer (0.1M sodium phosphate, 0.5M NaCl, pH 7.5). Monoclonal antibody(anti-glucose oxidase) (1.5 mg in 2.0 mL of coupling buffer) was addedto the fibrils and the suspension was rotated for 1 hour to allow theNHS fibrils to react with the antibody (primarily with lysine sidechainsof the antibody). The resulting fibrils were filtered and repeatedlywashed with coupling buffer.

Binding of Glucose Oxidase to Antibody-Modified Fibrils

Non-antibody coated sites on antibody-modified fibrils were blocked byrotation for 10 minutes with 1% BSA dissolved in phosphate-bufferedsaline solution, pH 6.0. Glucose oxidase was dissolved in the BSAsolution and this solution was mixed with the fibrils for 1.5 hours.Finally, the fibrils were washed with neutral pH phosphate buffer untilno protein was observed to elute by spectrophotometrically monitoringthe effluents at 280 nm.

Detection of Antibody on Antibody-Modified Fibrils

Functional antibody molecules on the fibrils were quantitated by thecatalytic activity of bound antigen, glucose oxidase. Glucose oxidaseactivity was spectrophotometrically-determined by adding a sample of theenzyme (either free enzyme or enzyme specifically bound toantibody-modified fibrils) to a cuvette containing 0.1M sodium phosphate(pH 6.0), 20 μM glucose, 183 nM horseradish peroxidase, and 30 μM2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS). Theactivity of glucose oxidase was measured by spectrophotometricallydetermining the rate of absorbance increase at 414 nm at 25° C.(formation of green color). Quantitation of the bound enzyme indicatedthat there was 1.52 μmoles of functional antibody per gram of fibrils.

Fibril-Antibody Coupling Between Primary Amine and Carbohydrate Groups

Formation of Amino Fibrils

A suspension of NHS-fibrils (214.7 mg) in freshly prepared 0.2M NaHCO₃,pH 8.1 was mixed with 1,2-diaminoethane (100 μL) at room temperature.The suspension was stirred for approximately 5 hours. The mixture wasthen suction filtered in a Buchner funnel and washed with water (3×10mL) and methanol (3×10 mL) and dried in vacuo overnight. The yield ofamino fibrils was 144 mg. Ninhydrin testing demonstrated thatdiaminoethane (non-conjugated) was still absorbed to the fibrils.Fibrils were resuspended in water and sonicated for 90 minutes.Ninhydrin testing indicated that the fibrils were sufficiently free ofnon-covalently bound amine.

Formation of Antibody-Modified Fibrils

A NAP-5 column was equilibrated with 100 mM acetate, pH 5.5 andmonoclonal antibody (anti-glucose oxidase, 1.5 mg in 300 μL buffer) wasadded and eluted with buffer (1 mL). This solution was allowed to reactwith 50 μL of 20 mM NaIO₄ (final NaIO₄ concentration of 1 mM) at roomtemperature for 2 hours. The reaction was terminated by addition to apre-equilibrated NAP-10 (0.15M KH₂ PO₄, pH 6.0) and eluted with the samebuffer. The eluent was collected in a 10-mL tube containing aminofibrils and the suspension was allowed to react for 2-3 hours. Thefibrils were then washed with reaction buffer and stored in 5 mL KH₂PO₄, pH 5.5.

Detection of Antibody on Antibody-Modified Fibrils

Functional antibody molecules on the fibrils were quantitated by thecatalytic activity of bound antigen, glucose oxidase as described above.Quantitation of the bound enzyme indicated that there was 0.36 μmolesfunctional antibody per gram of fibrils. ##STR20## Reduction of MouseMonoclonal Antibody

To an antibody solution (24.7 mg, in 2.9 ml) was added dithiothreitol(DTT, 25 mg, 0.16 mmol) and the mixture was incubated for 1 hour at roomtemperature. The reduced antibody was purified using a PD-10 disposablegel filtration column (Pharmacia).

Covalent Coupling of Antibody and Maleimide Fibrils

Maleimide fibrils (0.18 mg) and plain fibrils (0.15 mg) were sonicatedfor 15 minutes at room temperature and incubated for 30 minutes at 40°C. The fibrils were then centrifuged and the supernatant was removed.Fibrils were then incubated with reduced antibody (12.4 mg) in sodiumphosphate buffer (0.1M, pH 7.2, 1.1 ml) for 4 hours at room temperature.

Quantitation of Antibody on Antibody Fibrils Using HorseradishPeroxidase (HRP)-Labeled Goat Anti-Mouse Antibody

Mouse antibody fibrils were pre-incubated with 0.1% PEG in sodiumphosphate buffer (0.1M, pH 7.5, 1 ml) for 30 minutes at 40° C. They werethen centrifuged and the supernatant was removed. Mouse antibody fibrilswere incubated with HRP-labeled goat anti-mouse antibody (0.01mg) insodium phosphate buffer (0.1M, pH 7,5, 500 ml) containing 0.1% PEG for 2hours at room temperature. The fibrils were then washed five times with0.1% PEG in sodium phosphate buffer (0.1M, pH 7.5, 1 ml). The amount ofbound HRP was determined by spectrophotometrically measuring thecatalytic activity of the bound enzyme. The enzyme reaction is shownbelow: ##STR21##

The results showed that the density of the mouse antibody on the fibrilswas 1.84 mg antibody per gram of fibrils and the density of the mouseantibody on plain (control) fibrils was 0.48 mg antibody per gram offibrils (the extent of nonspecific binding was about 26%).

EXAMPLE 8 Preparation of Bifunctional Fibrils by Addition of Lysine

Synthesis of N.sub.α -CBZ-L-lysine benzyl ester:

The reaction sequence is shown in FIG. 5. N.sub.ε-(tert-butoxycarbonyl)-L-lysine (2 g, 8.12 mmol) was dissolved inmethanol (40 ml) and water (40 ml), and the pH was adjusted to 8 withtriethylamine. A solution of N-(benzyloxycarbonyl-oxy)succinimide indioxane (2.4 g, 9.7 mmol in 20 ml) was added to the above mixture andthe pH was maintained at 8-9 with triethylamine. The reaction mixturewas stirred overnight. The solvent was removed by rotary evaporation toobtain crude N.sub.α -CBZ-N.sub.ε -(tert-butoxycarbonyl)-L-lysine.N.sub.α -CBZ-N.sub.ε -(tert-butoxycarbonyl)-L-lysine was treated with0.2M calcium carbonate (4 ml) and the aqueous layer was removed toobtain a white solid. The solid was resuspended inN,N-dimethylformamide(40 ml) and benzyl bromide (1.16 ml). The reactionmixture was stirred overnight at room temperature. The reaction mixturewas worked up with ethyl acetate and water, and the organic layer wasdried over magnesium sulphate. The solvent was removed to obtain crudeN.sub.α -CBZ-N.sub.ε -(tert-butoxycarbonyl)-L-lysine benzyl ester whichwas purified by silica gel chromatography using 25% hexane in ethylacetate as a solvent. To N.sub.α -CBZ-N.sub.ε-(tert-butoxycarbonyl)-L-lysine benzyl ester (1 g, 2.2 mmol) inmethylene chloride (10 ml) was added trifloroacetic acid at 0° C. Thereaction mixture was stirred for 10 minutes at 0° C., then stirred forfurther 2.5 hr at room temperature. The solvent was removed and thecrude product was obtained. Pure N.sub.α -CBZ-L-lysine benzyl ester wasobtained by silica gel chromatography.

Synthesis of N.sub.α -CBZ-L-lysine benzyl ester fibrils:

To a suspension of carboxyl fibrils (300 mg) in methylene chloride (18ml) was added a solution of N.sub.α -CBZ-L-lysine benzyl ester (148 mg,0.32 mmol in 20 ml methylene chloride and 176 μl triethylamine). HOBT(43.3 mg, 0.32 mmol) and EDC (61.3 mg, 0.32 mmol) were then added. Thereaction mixture was stirred overnight at room temperature to obtain thecrude product. The product fibrils were extensively washed withmethanol, methylene chloride, and water, then dried under vacuum.

Synthesis of bifunctional fibrils Fib-Lys(COOH)NH₂ :

To N.sub.α -CBZ-L-lysine benzyl ester fibrils (113 mg) in methanol (4ml) was added sodium hydroxide (1 N, 4 ml) and the reaction mixture wasstirred overnight. The product N.sub.α -CBZ-L-lysine fibrils wasextensively washed with water and methanol and the fibrils were driedunder vacuum. To a suspension of N.sub.α -CBZ-L-lysine fibrils (50 mg)in acetonitrile (4 ml) was added trimethyl silyl iodide (1 ml). Themixture was stirred for 3 hours at 40° C. The final bifunctional fibrilswere extensively washed with water, methanol, 0.5N sodium hydroxide,acetonitrile and methylene chloride. Amino acid analysis showed 0.3μmols lysine per gram of fibrils.

Hydroxyl and carboxyl (or amino) bifunctional fibrils can be made by asimilar method to that described here by using serine, threonine, ortyrosine. Thiolated and carboxyl (or amino) bifunctional fibrils can bemade using cysteine. Carboxyl and amino bifunctional fibrils can be madeusing aspartic or glutamic acid.

EXAMPLE 9 Carbon Nanotube as ECL-Based Biosensors

To a suspension of bifunctional fibril (113 mg) in methylene chloride (8ml) were added 4-dimethylaminopyridine (DMAP, 19.5 mg, 0.16 mmol) and1,3 dicyclohexylcarbodiimide (DCC, 33 mg, 0.16 mmol) (FIG. 6). Themixture was stirred for 5 minutes, then 4-methyl-4'-(8-hydroxyoctyl)2,2'-bipyridine (47 mg, 0.16 mmol) was added. The reaction mixture wasstirred overnight at room temperature. The resulting product fibrilswere extensively washed sequentially with DMF, 50% dioxane in water,methanol, and water. The fibrils were suspended in a mixture of ethanol(4 ml) and water (4 ml) and cis-dichlorobis (2,2'-bipyridine) ruthenium(II) dihydrate (45.2 mg, 0.087 mmol) was added. The mixture was refluxedfor 5.5 hours at 110° C. The ruthenium complex-modified fibrils wereextensively washed with water, standard ECL assay buffer (IGEN, Inc.,Gaithersburg, Md.), toluene, 50% dioxane in water, then sequentiallyrefluxed in acetonitrile, ethylene glycol and methanol. The rutheniumcomplex-modified fibrils were reacted with TMSI (4 ml) in acetonitrile(4 ml) for 4 hours at 40° C. to deprotect the CBZ group, then washedwith methanol, water, and sodium hydroxide (1N). The final produce wasdried under vacuum. The fibrils were then suspended in methylenechloride (5 ml) and triethylamine (5 drops) was added. To the suspensionwas added succinic anhydride (40 mg). The reaction mixture was stirredovernight at room temperature and the product was washed with methylenechloride, methanol, and water, then dried under vacuum. The carboxylicacid/ruthenium complex-modified fibrils were resuspended in dioxane (5ml), then N-hydroxysuccinimide (100 mg) and EDC (167 mg) were added. Thereaction mixture was stirred for 4 hours at room temperature. Theresulting NHS ester/ruthenium complex-modified fibrils were washed withdioxane and methanol. The NHS ester/ruthenium complex-modified fibrilswere resuspended in dioxane (2 ml) and a solution of NAD analog insodium bicarbonate (75 ml in 2 ml of 0.2M pH 8.6 NaHCO₃) was added. Thereaction mixture was stirred overnight at room temperature. The fibrilswere then extensively washed with water, sodium bicarbonate (0.2M), andmethanol, then dried under the vacuum to obtain the biosensor fibrils.

EXAMPLE 10 Use of Carbon Nanotubes as ECL-Based Biosensors

An ECL-based biosensor was prepared by chemical modification of fibrils.The modified fibrils were prepared using NH₂ /COOH bifunctional fibrils(Example 8). To one functional group (NH₂) was added an NAD⁺ analog andto the other functional group (COOH) was added a derivative of Ru(bpy)₃²⁺. The structure of the biosensor fibrils is shown in FIG. 7.

The biosensor was designed to facilitate detection of dehydrogenaseenzymes that accept NAD(P)⁺ /NAD(P)H as a cofactor and substrates ofdehydrogenase enzymes that accept NAD(P)⁺ /NAD(P)H as a cofactor. It isknown (E. Jameison et al., Analytical Chemistry, in press) that NAD(P)⁺and NAD(P)H have dramatically different abilities to promote Ru(bpy)₃ ²⁺ECL. Thus, the activity of a dehydrogenase can be detected by itsability to reduce/oxidize NAD(P)⁺ /NAD(P)H, which is observable bydifferences in the abilities of NAD(P)⁺ and NAD(P)H to cause Ru(bpy)₃ ²⁺electrochemiluminescence (FIG. 8). Similarly, because the action ofdehydrogenases on their substrates is stoichiometrically accompanied byconversion of NAD(P)⁺ to NAD(P)H or NAD(P)H to NAD(P)⁺, the presence oftheir substrates can also be detected by electrochemiluminescence.

To use the fibril supported ECL-based biosensor, the biosensor is mixedwith an aqueous solution containing a dehydrogenase and an unknownquantity of the dehydrogenase's substrate (the substrate is the analyte)or an aqueous solution containing a dehydrogenase substrate and anunknown quantity of dehydrogenase (the dehydrogenase is the analyte).After a suitable incubation time to allow the enzyme reaction to proceedand the NAD(P)⁺ or NAD(P)H immobilized on the fibrils to be reduced oroxidized, the fibrils are drawn into an ECL instrument and the ECL ofthe fibrils is measured. ECL measurement is carried out in a buffer thatdoes not contain appreciable concentrations of tripropylamine (becauseNAD(P)⁺ /NAD(P)H are tripropylamine replacements in this invention).

The attractive features of this ECL-based biosensor are: the closeproximity of NAD(P)⁺ /NAD(P)H and Ru(bpy)₃ ²⁺ on the same bifunctionalgroup on the fibrils which enhances the efficiency of electron transferin the electrochemical ECL mechanism (intramolecular electron transferis more efficient than intermolecular electron transfer); the ECL activereagents, NAD(P)⁺ /NAD(P)H and Ru(bpy)₃ ²⁺ are both supported on fibrilswhich can be magnetically held on the ECL instrument electrode whichwill increase light emission; fibrils have extremely high surface areaswhich permit immobilization of a high density (per weight) of NAD(P)⁺/NAD(P)H and Ru(bpy)₃ ²⁺ which will allow more light to be emitted; thebiosensor is versatile in that it can detect many different analytes(any dehydrogenase that accepts NAD(P)⁺ or NAD(P)H as a cofactor, or thesubstrates of these enzymes).

EXAMPLE 11 Detection of glucose-6-phosphate dehydrogenase (G6PDH) usinga fibril supported ECL-based biosensor

Fibrils modified with both an NAD⁺ analog and a Ru(bpy)₃ ²⁺ analog beingcovalently attached to a bifunctional adduct (ECL biosensor fibrils)were used to detect the enzyme glucose-6-phosphate dehydrogenase. ECLbiosensor fibrils (860 μg/mL) were mixed with a neutral pH bufferedsolution containing approximately 50 μM glucose-6-phosphate. Into onetube was added G6PDH to a concentration of 3.6 μM. Into a second controltube was added deionized water. Immediately the ECL of the fibrils wasmeasured. FIG. 9 shows that at time zero the ECL of the fibrils with(DH+) and without (DH-) G6PDH was a similar level. The background ECLresulting from the assay buffer (no fibrils) is also shown (A.B.). After42 hours of incubation (rotation at room temperature) more fibrils werewithdrawn and the ECL was measured again. As shown in FIG. 9, the ECL ofassay buffer (A.B.) and fibrils in the absence of G6PDH (DH-) wassimilar to the ECL seen at time zero. However, the ECL of the samplecontaining G6PDH (DH+) was substantially lower than at time zero. Theresults indicate that G6PDH activity could be detected using fibrilsupported ECL biosensors. Separate work with the particular NAD⁺ analogused here (non-immobilized) and Ru(bpy)₃ ²⁺ (non-immobilized) in theabsence of fibrils confirmed that the reduced (NADH) version of the NAD⁺analog is less efficient than the oxidized (NAD⁺) version at causingRu(bpy)₃ ²⁺ to be electrochemiluminescent.

EXAMPLE 12 Use of Carbon Nanotubes as a Support for an ECL-Based EnzymeBiosensor

An enzyme biosensor was prepared containing a dehydrogenase enzyme towhich has been conjugated both an analog of a nicotinamide adeninedinucleotide (NAD⁺) enzyme cofactor and an analog of ruthenium (II)trisbipyridine (Ru(bpy)₃ ²⁺). The NAD⁺ analog is tethered so that it canbind in the enzyme's cofactor binding site and behave naturally whenbound (i.e., it can be reduced by the enzyme by the normal chemicalmechanism in the presence of the natural substrate of the enzyme).Moreover, the Ru(bpy)₃ ²⁺ analog is tethered so that it can come intophysical contact with the NAD⁺ (FIG. 10). In an ECL instrument such asan IGEN Origen® Analyzer (IGEN, Inc., Gaithersburg, Md.), NAD⁺ and itsreduced form, NADH, promote Ru(bpy)₃ ²⁺ electrochemiluminescence todifferent extents. Thus, based on the light output, it can be determinedwhether NAD⁺, NADH, or some mixture of the two is present in a solution(F. Jameison et al., Analytical Chemistry, in press). Thus, thebiosensor uses the differences between the efficiencies of the ECLreactions of Ru(bpy)₃ ⁺ to detect the extent of reduction of NAD⁺ andhence the presence of the enzyme's substrate. As an example, alcoholdehydrogenase catalyzes the reaction shown below; ethanol (reduced)+NAD⁺(oxidized)→ acetaldehyde (oxidized)+NADH (reduced). An alcoholdehydrogenase-based ECL biosensor can convert ethanol to acetaldehyde,concomitantly converting NAD⁺ to NADH. Because the ECL properties ofRu(bpy)₃ ²⁺ immobilized on the biosensor depend on whether NAD⁺ or NADHis immobilized, the ECL of the enzyme biosensor can report the presenceof ethanol. An attractive feature of the biosensor is that becauseduring the ECL reaction the NADH form of the cofactor is re-oxidized tothe NAD⁺ form, one biosensor molecule can be used repeatedly to detectmultiple molecules of analyte.

Because the biosensor is based on a soluble enzyme (a dehydrogenase) andthe analyte (ethanol for example) is also soluble, it would bebeneficial to immobilize the biosensor so that it could be usedrepetitively to analyze different analyte-containing samples withoutbeing washed away with a spent analyte-containing solution. Suchimmobilization was carried out with an alcohol dehydrogenase (ADH) basedECL biosensor (shown schematically in FIG. 10). FIG. 11 showsimmobilization of the ADH-based biosensor on Dynal M450 beads (LakeSuccess, N.Y.) (left) and immobilization on alkyl fibrils (right).Neither diagram is drawn to scale such that the biosensor molecules areactually much smaller relative to the size of the solid supports. Thealkyl fibrils were prepared by the reaction of oxidized fibrils (bearingCOOH groups) with 1-aminooctane. In both cases (beads and fibrils) theenzyme biosensor was immobilized by noncovalent adsorption in bufferedaqueous solution.

Ethanol was detected using both bead- and fibril-immobilized ECLADH-based biosensors. The amount of enzyme biosensor adsorbed to beadsor fibrils was determined by measuring the amount of remaining(non-absorbed) protein in solution by the UV absorbance at 280 nm (theabsorbance at 280 nm indicates the amount of protein in solution). Theresults showed that 0.308 mg of the ADH biosensor bound per mg offibrils while 0.015 mg of the ADH biosensor bound per mg of beads. Thus,per unit weight of the solid support, the absorptive capacity of fibrilsexceed that of Dynal beads by 20.5 times.

ECL sensing of ethanol was carried out by mixing the ADH biosensoradsorbed beads (0.50-1.25 mg) or fibrils (0.04-0.10 mg) with a solutioncontaining 0.1M sodium phosphate buffer (pH 7.2), 12 mM semicarbizide.Some samples also contained 0.5 mM of the analyte, ethanol. The solidsupports coated with the biosensor were drawn into an IGEN Origen® ECLAnalyzer (IGEN, Inc., Gaithersburg, Md.) and ECL was measured. Resultsof one such experiment are shown in FIG. 12. With both beads andfibrils, the ECL signal of the enzyme biosensor decreased in thepresence of ethanol. This result is consistent with results obtained insolution studies (using non-immobilized NAD⁺ /NADH and non-immobilizedRu(bpy)₃ ²⁺) of the effect of the oxidation/reduction state of this NAD⁺analog on Ru(bpy)₃ ²⁺ electrochemiluminescence. These results showedthat fibrils can be used as a support for enzymes in ECL and inparticular as a support for enzyme-based ECL biosensors. It should alsobe noted that the results seen with fibrils were similar to those seenwith Dynal beads even though 20 times less fibrils was used.

EXAMPLE 13 Biotinylated Fibrils and Bifunctional Biotinylated AlkylFibrils

It has been found that fibril surfaces can be biotinylated or bothalkylated and biotinylated. The fibrils containing such modificationscan then bind any streptavidin conjugated substances such asstreptavidin beads and streptavidin enzymes.

Fibrils offer great advantages as solid carriers because of their highsurface area. Beads, which can be made strongly magnetic, are extremelyuseful in separation assays. The biotinylated fibrils described hereincombine the advantages of both the fibrils and the beads. Thebiotinylated alkyl fibrils are an extension of the same concept butexhibit the additional protein adsorption property of alkyl fibrils.

The streptavidin- and biotin-coated fibrils can be used in diagnosticsand can be used as capture agents for electrochemiluminescence assays.

A novel feature of this invention is the combination of two solidcarriers on one fibril to create a bifunctional fibril. Moreover, thedisclosed process increases the surface area for beads and magnifiesfibril magnetization.

Preparation of Biotinylated Fibrils

Biotinylated fibrils were prepared by mixing 2.4 mg of amino fibrilsprepared essentially as described in Preparation O and 9 mg of NHS esterlong chain biotin in buffer 0.2M NaHCO₃ at a pH of 8.15. The mixture wasrotated at room temperature for four hours and washed with the samebuffer twice.

Preparation of Biotinylated Alkyl Fibrils

Biotinylated alkyl fibrils were prepared by a two step reaction. First,4.25 mg of bifunctional fibrils (containing both amino and carboxyl) and25 mg of NHS ester long chain biotin were mixed. The fibrils were washedand dried under vacuum.

The second reaction was carried out by mixing 4 mg of biotinylatedbifunctional fibrils with 11 mg of EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), 7.5 mg of DMAP(4-dimethylaminopyridine) and 10 μl of NH₂ (CH₂)₇ CH₃ in 0.5 ml of DMF.The mixture was stirred at room temperature overnight. The finalbiotinylated alkyl fibrils were washed by CH₂ Cl₂, MeOH, and dH₂ O.

EXAMPLE 14 Biotinylated Fibrils as a Solid Support in ECL Assays

Biotinylated fibrils can be used in ECL assays involving formats thatrequire streptavidin-biotin or avidin-biotin interactions. Biotinylatedfibrils could for example be further derivatized with streptavidin.Biotin covalently linked to fibrils (see Example 13) could form strongnon-covalent binding interactions with streptavidin. Becausestreptavidin is a tetrameric protein with four equivalent binding sites,streptavidin bound to biotinylated fibrils would almost certainly haveunoccupied binding sites to which additional biotinylated reagents couldbind. Thus, biotinylated fibrils would be converted tostreptavidin-coated fibrils.

There are a number of analytical tests that could be performed with suchfibril-biotin-streptavidin (FBS) supports. For example, a biotinylatedanti-analyte antibody could be captured on the FBS support (eitherbefore or after the antibody has complexed to an analyte). Assays usingbiotinylated anti-analyte antibodies are well established. Such assaysinclude competitive assays where the analyte of interest competes withan introduced Ru(bpy)₃ ²⁺ -labeled analyte for binding to theanti-analyte antibody. Free (unbound) analyte and free (unbound)Ru(bpy)₃ ²⁺ -labeled analyte can be washed from the fibril immobilizedantibody. The washing step depends on the fibrils being physicallyseparated from the solution phase by common practices involvingcentrifugation, filtration, or by attraction to a magnet.

Besides a competition assay, a sandwich type immunoassay could becarried out on FBS supports. Sandwich immunoassays are well known in thefield of diagnostics in general and in ECL detection in specific. Suchassays involve an analyte being bound simultaneously by two antibodies;a first "primary" antibody which is captured on a solid surface by forexample being labeled with biotin, and a "secondary" antibody which isnot captured by a solid surface but is labeled with a reporter group,such as Ru(bpy)₃ ²⁺ in ECL applications. Such a sandwich assay could becarried out using fibrils as a solid capture support whereby the fibrilsare captured as described in the previous paragraph. Hence, in such anassay, the fibril would have covalently linked to it biotin, which wouldbe bound to streptavidin, which would in turn be bound to a biotinylatedprimary antibody, which would be bound to analyte (if present), whichwould be bound to a Ru(bpy)₃ ²⁺ -labeled secondary antibody.

Similarly, DNA probe assays could be carried out using FBS supports.Biotinylated single stranded DNA can be bound to FBS supports andcompetitive hybridization can occur between complementary singlestranded analyte DNA molecules and complementary Ru(bpy)₃ ²⁺ -labeledoligonucleotides.

Another type of biotinylated fibrils, biotinylated alkylated fibrils,can be used in immunoassays and DNA probe assays. As described inExample 13, bifunctional fibrils can be modified by covalent attachmentof biotin to one type of functional group and alkyl chains to the othertype of functional group. The resultant alkylated, biotinylated fibrilscan be used both in specific association with streptavidin or avidin(via biotin) and also for adsorption of proteins (via the alkyl chains).

Alkyl fibrils could be used in conjuction with other solid supports,such as streptavidin-coated magnetic beads, including Dynal magneticbeads. One advantage of fibrils over such beads is that they have a muchhigher surface area (per unit weight). Thus, if fibrils could beattached to the outside surface of the magnetic beads, this woulddramatically improve the surface area and hence the binding capacity ofthe beads. It is envisioned that alkylated, biotinylated fibrils couldbe mixed with streptavidin-coated beads resulting in high affinitystreptavidin(bead)-biotin(fibril) interactions and hence fibril-coatedbeads with an extremely high surface area. Because alkyl fibrils canbind proteins by adsorption, the fibril-coated beads could be furtherderivatized with adsorbed proteins including streptavidin andantibodies. As described above, streptavidin or antibody coated fibrilscan be used in immunoassays and DNA probe assays. Thus, fibril-coatedbeads could improve the properties of the beads by dramaticallyincreasing their surface area such that fewer beads would be required ina given assay to give the same result.

EXAMPLE 15 Magnetically-Susceptible Carbon Nanotubes

Fibrils may have magnetic properties. The extent to which fibrils can bemade magnetic or nonmagnetic is controlled by the amount of catalystthat is in the fibril as a result of the fibril production process. Suchprocesses are disclosed in U.S. Pat. Nos. 4,663,230, 5,165,909 and5,171,560.

The magnetic property of fibrils was observed after fibrils werefunctionalized, i.e. alkyl fibrils, protein bound fibrils, etc. Thefibrils after certain reactions (e.g., alkylation) migrated notablyfaster to a magnet than before the reaction. One hypothesis for thisphenomena is that some kind of aggregation or salvation may be occurringduring the reaction process. However, the true mechanism is presentlyundetermined.

EXAMPLE 16 Reduction of Non-specific binding of Ru(Bpy)₃ with PEGmodified Fibrils

Stock dispersions of chlorate oxidized fibrils, fibrils modified withPEG using benzoyl perioxide and fibrils modified with PEG by NHS estercoupling were prepared at 1.0 mg/ml in 50 mM potassium phosphate buffer,pH 7.0 with sonication. 2 mls of 10-fold serial dilutions of each wereplaced in each of 5 polypropylene tubes. 50 μl of a stock solution ofRu(Bpy)₃ (approx. 10 μM) in the same buffer was added to each tube andto 3 buffer blanks. Three bufffer tubes without Ru(Bpy)₃ were alsoprepared. All tubes were mixed on a vortex mixer and allowed to incubateovernight. All tubes were centrifuged to separate the fibrils and 0.1 mlaliquots of the supernatant were transfered to new tubes and dilutedwith 1.0 mls of Origen® Assay Buffer (IGEN, Inc., Gaithersburg, Md.) andanalyzed for Ru(Bpy)₃ by ECL using a Magnalyzers (IGEN, Inc.,Gaithersburg, Md.). The level of Ru(Bpy)₃ remaining in the supernatantwas an indirect measure of the amount that had been non-specificallybound to the fibrils (FIG. 13). For both of the PEG modified fibrilmaterials substantially all of the Ru(Bpy)₃ remained in the supernatantat fibril levels up to 0.1 mg/ml. There was 20-30% decrease in theRu(Bpy)₃ in the supernatant at 1.0 mgs/ml of these fibrils. In contrast,for the chlorate oxidized fibrils, there was almost no Ru(Bpy)₃remaining in the supernatant at 1.0 mgs/ml and 20-30% decrease in theRu(Bpy)₃ in the supernatant at 0.1 mg/ml of these fibrils without thePEG modification.

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What is claimed is:
 1. A nanotube comprising carbon to which is attachedan assay-performance substance, said assay-performance also being linkedto a label compound capable of being induced to luminesce.
 2. A nanotubeaccording to claim 1, wherein said nanotube is graphitic and saidluminescence is electrochemiluminescence.
 3. A composition for thedetection of an analyte of interest present in a sample, whichcomposition comprises:(i) a graphitic nanotube containing a functionalgroup and (ii) an assay-performance-substance linked to said functionalgroup, said assay-performance-substance being capable of binding to theanalyte-of-interest.
 4. A composition for the detection of an analyte ofinterest present in a sample, which composition comprises:(i) agraphitic nanotube containing a functional group and (ii) anassay-performance-substance linked to said functional group, saidassay-performance-substance being bound to the analyte of interest.
 5. Acomposition according to claim 4, further comprising a secondassay-performance-substance bound to the analyte, said secondassay-performance-substance being linked to a label compound capable ofbeing induced to luminesce.
 6. A composition for the detection of ananalyte of interest present in a sample, which composition comprises(a)a graphitic nanotube containing a functional group and (b) anassay-performance-substance linked to said functional group wherein saidassay-performance-substance contains at least one substance selectedfrom the group consisting of(i) added analyte of interest or addedanalogue of said analyte; (ii) a binding partner of said analyte or abinding partner of an analogue of said analyte; and (iii) a reactivecomponent capable of binding with (i) or (ii).
 7. A compositionaccording to claim 5, wherein said assay-performance-substance containsat least one substance selected from the group consisting of(i) addedanalyte of interest or added analogue of said analyte; (ii) a bindingpartner of said analyte or a binding partner of said analogue; and (iii)a reactive component capable of binding with (i) or (ii).
 8. A methodfor performing a binding assay for an analyte of interest present in asample comprising the steps of:(a) forming a composition containing(i)said sample (ii) a first assay-performance-substance linked to a labelcompound capable of being induced to luminesce, and (iii) a plurality offunctionalized graphitic nanotubes bound to a secondassay-performance-substance; (b) incubating said composition to form acomplex which includes said graphitic nanotube bound to said secondassay-performance-substance and said label compound bound to said firstassay-performance-substance; (c) collecting said complex in ameasurement zone; (d) inducing the label compound in said complex toluminesce, and (e) measuring the emitted luminescence to measure thepresence of the analyte of interest in the sample.
 9. A method asrecited in claim 8 wherein said complex is collected on the surface ofmeans for inducing luminescence and measuring said luminescence.
 10. Amethod as recited in claim 9 based upon measurement ofelectrochemiluminescence wherein said complex is collected at anelectrode surface.
 11. A method for performing a binding assay for ananalyte of interest present in a sample based upon measurement ofelectrochemiluminescence at an electrode surface comprising thesteps:(a) forming a composition containing(i) said sample (ii) a firstassay-performance-substance linked to a label compound capable of beinginduced to electrochemiluminesce, and (iii) a plurality offunctionalized graphitic nanotubes bound to a secondassay-performance-substance; (b) incubating said composition to form acomplex which includes said graphitic nanotube bound to said secondassay-performance-substance and said label compound bound to said firstassay-performance-substance; (c) collecting said complex; (d) causingsaid collected complex to come in contact with an electrode surface andinducing the label compound in said complex to luminesce by impressing avoltage on said electrode; and (e) measuring the emitted luminescence atthe electrode surface to measure the presence of the analyte of interestin the sample.
 12. A method for performing a binding assay for ananalyte of interest present in a sample based upon measurement ofelectrochemiluminescence at an electrode surface comprising thesteps:(a) forming a composition containing(i) said sample (ii) A firstassay-performance-substance linked to a label compound capable of beinginduced to electrochemiluminesce, and (iii) a plurality of magneticallyresponsive suspended graphitic nanotubes bound to a secondassay-performance-substance; (b) incubating said composition to form acomplex which includes a graphitic nanotube and said label compound; (c)collecting said complex by imposition of a magnetic field on saidgraphitic nanotubes; (d) causing said collected complex to come incontact with an electrode surface and inducing the label compound insaid complex to luminescence by imposing a voltage on said electrode;and (e) measuring the emitted luminescence at the electrode surface tomeasure the presence of the analyte of interest in the sample.
 13. Amethod as recited in claim 12 wherein the imposition of said magneticfield causes said complex to collect at the surface of said electrode.14. A composition of matter for use as a reagent in amicroparticulate-based binding assay comprising functionalized graphiticnanotubes bound to an assay-performance-substance and at least one othercomponent selected from the group consisting of:(a) electrolyte; (b)label compound containing an ECL moiety; (c) analyte of interest or ananalog of the analyte of interest; (d) a binding partner of the analyteof interest or of its analog; (e) a reactive component capable ofreacting with (c) or (d); (f) a reductant; and (g) anelectrochemiluminescent-reaction enhancer; provided, however, that notwo components contained within any reagent composition are reactivewith one another during storage so as to impair their function in theintended assay.
 15. A reagent as recited in claim 14 containingmagnetically responsive graphitic nanotubes.
 16. An assay reagent for anassay based upon a binding reaction and the measurement of anelectrochemiluminescent phenomenon comprising:(a) an electrolyte; (b) aplurality of magnetically responsive functionalized graphitic nanotubesbound to a first assay-performance-substance; and (c) a secondassay-performance-substance bound to a chemical moiety havingelectrochemiluminescent properties.
 17. A method for performing an assayfor an analyte of interest present in a sample, which analyte is anenzyme, comprising the steps of:(a) forming a composition containing(i)said sample, and (ii) a functionalized graphitic nanotube bound to acomponent which is bound to a label compound capable of being induced toluminesce, wherein said component is a substrate of the analyte ofinterest; (b) incubating said composition under conditions to permitsaid analyte to cleave said component, whereby a label sub-componentfree from said nanotube results from said cleavage; (c) separating saidgraphitic nanotube from said composition; (d) inducing the free labelsub-component to luminesce; and (e) measuring the emitted luminescenceto measure the presence of the analyte of interest in the sample.
 18. Agraphitic nanotube linked to at least two components selected from thegroup consisting of (i) an electrochemiluminescent label, (ii) an enzymeand (iii) an enzyme cofactor.
 19. A graphitic nanotube linked to anenzyme biosensor, wherein said enzyme biosensor includes a labelcompound capable of being induced to electrochemiluminesce.